Inactivatable target capture oligomers for use in the selective hybridization and capture of target nucleic acid sequences

ABSTRACT

The present invention provides compositions, kits and methods for the selective hybridization and capture of a specific target nucleic acid. The specific target nucleic acid may be present in a heterogeneous mixture of nucleic acids. Selective hybridization and capture provides a target nucleic acid that is substantially free of non-target and/or contaminating nucleic acids. Target nucleic acids that have been selectively hybridized and captured using the current invention are then used in subsequent analysis, wherein the presence of non-target and/or contaminating nucleic acids that interfere with said subsequent analysis have been substantially reduced or eliminated, thereby providing improved analysis results. The invention offers the further advantage of requiring less stringent purification and/or sterility efforts than conventionally needed in order to ensure that enzymes and other reagents used in subsequent analysis, or present in the environment in which an assay is performed, are free of bacterial or other contaminating nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.provisional application Ser. No. 61/052,944, filed May 13, 2008.

FIELD OF THE INVENTION

This invention relates to methods, compositions, reaction mixtures andkits for the selective hybridization of multiple copies of a specifictarget nucleic acid sequence, which may be present either alone or as acomponent of a homogeneous or heterogeneous mixture of nucleic acids.Selectively hybridized target sequences are isolated for subsequentprocessing, such as analysis and/or storage of said target sequence.Analysis preferably includes amplification and detection of the targetnucleic acid.

DESCRIPTION OF THE RELATED ART

The isolation and purification of nucleic acid molecules is an importantstep for a variety of downstream procedures such as nucleic analysis,nucleic acid reagent preparation, bulk drug substance preparation andthe like. Desired for the isolation step is a bias of the selectionmedium towards the desired nucleic acid and away from contaminatingnucleic acids. The objective is to maximize recovery of the desirednucleic acid and minimize the presence of contaminating nucleic acids.Nucleic acid isolation is currently performed using a variety oftechniques, including attachment to solid supports.

Attachment of nucleic acid molecules to solid supports is generallyknown in that art. In U.S. Pat. No. 5,599,667, Arnold et al. describethe use of polycationic solid supports for selective capture andimmobilization of large nucleic acids from mixtures of large and smallnucleic acids. The immobilized nucleic acids are then detected.Similarly, U.S. Pub. No. 2008-0319182 describes a method for bindingnucleic acids to a solid support in the presence of imidizoles andguanidiniums. See also, U.S. Pub. No. 2006-0252085.

In U.S. Pat. No. 6,110,678, Weisberg et al. describe a two-stephybridization and capture of polynucleotides in a sample usingimmobilized probes attached to solid supports. In a first step, a targetcapture probe is hybridized with a target nucleic acid. In a secondstep, the target nucleic acid and target capture probe complex arehybridized to the immobilized probe of the solid support. Weisburg'stwo-step hybridization is a target specific method in that a selecttarget is captured by the target capture probe but the unwanted othernucleic acids in the sample are not. Weisburg's target capture probecomprises a first nucleic acid region that is configured to besubstantially complementary to a desired target nucleic acid and tospecifically hybridize to that target nucleic acid and not to othercontaminating nucleic acids suspected of being in a sample.

Capture probes and solid supports are used to isolate nucleic acids fromsamples for a variety of subsequent downstream analysis procedures.(See, for example, U.S. Pub. No. 2008-0199863 by Luo et al.; U.S. Pub.No. 2007-0003937 by Wang, U.S. Pub. No. 2005-0059024 by Conrad, U.S.Pub. No. 2006-0263769 by Haake et al, and U.S. Pat. No. 2008-0300142 byGetts et al.) Once isolated, a target nucleic acid is then useful for avariety of downstream analyses, such as, characterization of a targetnucleic acid molecule isolated from a cellular lysate or amplificationand detection of a target nucleic acid isolated from a diagnosticsample.

Detection and/or quantitation of specific nucleic acid sequences is animportant technique for identifying and classifying microorganisms,diagnosing infectious diseases, measuring response to various types oftreatment, and the like. Such procedures are also useful in detectingand quantitating microorganisms in foodstuffs, water, beverages,industrial and environmental samples, seed stocks, and other types ofmaterial where the presence of specific microorganisms may need to bemonitored. Numerous amplification-based methods for the detection andquantitation of target nucleic acids are well known and established inthe art. PCR uses multiple cycles of denaturation, annealing of primerpairs to opposite strands, and primer extension to exponentiallyincrease copy numbers of the target sequence (e.g., Mullis et al., U.S.Pat. Nos. 4,683,195 and 4,800,159; Mullis, U.S. Pat. No. 4,683,202;Gelfand et al., U.S. Pat. No. 5,804,375; Mullis et al. (1987) Meth.Enzymol. 155, 335-350; and Murakawa et al. (1988) DNA 7, 287-295). InRT-PCR, reverse transcriptase makes a complementary DNA (cDNA) from RNA,and the cDNA is then amplified by PCR to produce multiple copies of DNA(Gelfand et al., U.S. Pat. Nos. 5,322,770 and 5,310,652). Otherwell-known amplification methods include strand displacementamplification, (SDA), (e.g., Walker, et al. (1992), Proc. Natl. Acad.Sci. USA 89, 392-396; Walker et al., U.S. Pat. No. 5,270,184; Walker,U.S. Pat. No. 5,455,166; and Walker et al. (1992) Nucleic Acids Research20, 1691-1696); thermophilic SDA (tSDA), using thermophilicendonucleases and polymerases at higher temperatures in essentially thesame method as SDA (European Pat. No. 0 684 315); rolling circleamplification (RCA) (e.g., Lizardi, U.S. Pat. No. 5,854,033); helicasedependent amplification (HDA) (e.g., Kong et al., U.S. Pub. No. US2004-0058378 A1); loop-mediated isothermal amplification (LAMP) (e.g.,Notomi et al., U.S. Pat. No. 6,410,278); and transcription-basedamplification methods (e.g., Kwoh, D. et al. (1989) Proc. Natl. Acad.Sci. USA 86, 1173-1177), for example, NASBA (e.g., Malek et al., U.S.Pat. No. 5,130,238), Q.beta. replicase (e.g., Lizardi, P. et al. (1988)BioTechnol. 6, 1197-1202), self-sustained sequence replication (e.g.,Guatelli, J. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874-1878;Landgren (1993) Trends in Genetics 9, 199-202; and Helen H. Lee et al.,Nucleic Acid Amplification Technologies (1997)), andtranscription-mediated amplification, (TMA) (e.g., Kacian et al., U.S.Pat. Nos. 5,480,784 and 5,399,491). TMA synthesizes multiple copies of atarget nucleic acid sequence autocatalytically under conditions ofsubstantially constant temperature, ionic strength, and pH, in whichmultiple RNA copies of the target sequence autocatalytically generateadditional copies. TMA is a robust and highly sensitive amplificationsystem with demonstrated efficacy, and which overcomes many of theproblems associated with PCR-based amplification systems. In particular,temperature cycling is not required.

Amplification assays are particularly well suited for the detection ofmicroorganisms in the context of clinical laboratory testing, bioprocessmonitoring, or any other setting in which the detection ofmicroorganisms in a particular sample type is desired, by offering highsensitivity and rapid time-to-result relative to conventionalmicrobiological techniques. In addition, amplification methods can beused in the detection of the vast number of microorganisms that aredifficult or impossible to culture on synthetic media. Nevertheless,there are certain limitations associated with first-generationamplification assays that have limited their acceptance in certainsettings, such as clinical microbiological laboratories. One inherentproblem associated with the high sensitivity of nucleic acidamplification systems is that contaminating nucleic acid introduced intothe amplification system (e.g., from one or more reagents used duringamplification, from the technician performing the assay, from theenvironment in which the amplification is performed, etc.) can result infalse positive results. For example, even extremely small amounts ofnucleic acid contamination present in reagents and/or enzymes used in anamplification reaction, or in the environment in which the amplificationreaction is performed, can give rise to a positive amplification signaldespite the fact that the sequence of interest is not present in thenucleic acid sample being tested. This requires that significant effortbe expended in sample preparation, purification, sterilization, etc., ofthe reagents used in amplification reactions to avoid or minimize falsepositive results.

Accordingly, there is a need in the art for compositions and methods forisolating a target nucleic acid compound while minimizing the presenceof contaminating nucleic acids. There is also a need in the art forcompositions and methods allowing for a robust nucleic acidamplification that selectively amplifies target nucleic acid sequencesof interest while reducing or eliminating false positive results arisingas a result of contaminating biological material, such as contaminatingnucleic acid. There also remains a need for amplification systems thathave reduced reagent purification and/or sterility requirements. Asdescribed further herein, the present invention meets these needs andoffers other related advantages.

SUMMARY OF THE INVENTION

This invention relates to compositions, kits and methods for theselective hybridization of multiple copies of a specific target nucleicacid sequence, which may be present either alone or as a component of ahomogeneous or heterogeneous mixture of nucleic acids. The inactivatabletarget capture oligomers of the current invention comprise at leastthree nucleic acid sequence regions: namely a target hybridizationregion; a tag-closing region; and a binding pair member region.Preferably, the binding pair member is a substantially homopolymericnucleic acid sequence. Preferably, the tag-closing region iscomplementary to a portion of the target hybridization region such thatthe tag-closing region and target hybridization region will hybridizetogether under a set of conditions, thereby forming a hairpin structureas illustrated in the FIGS. and described herein. These inactivatabletarget capture oligomers selectively hybridized to target nucleic acidsequences, which are then isolated for subsequent processing, such asanalysis and/or storage of said target sequence. Analysis preferablyincludes amplification and detection. These inactivatable target captureoligomers are capable of an active and an inactive configuration. In theactive configuration and under a first set of conditions, theinactivatable target capture oligomers hybridize to a desired targetnucleic acid. A second set of conditions is then met in the reactionmixture to inactivate the inactivatable target capture oligomer. Byplacing the inactivatable target capture oligomer into its inactiveconfiguration its target hybridization region is blocked from furtherhybridization. Thus, the inactive configuration substantially reducesnon-specific hybridization of the inactivatable target capture oligomerto a nucleic acid in the reaction mixture caused by, e.g., changes inthe stringency conditions within the reaction mixture, time, size of thetarget hybridization region sequence, nucleic acid sequence similaritybetween the desired target nucleic acid and other non-targeted orcontaminating nucleic acids in the mixture, and/or other events known toresult in non-specific hybridization. Once a target nucleic acid isselectively hybridized to an inactivatable target capture oligomer ofthe current invention, the target can be captured using a solid supportcomprising a complementary binding pair member. Capture and washtechniques are well known in the art. (Weisburg et al., U.S. Pat. No.6,534,273). Selectively hybridized and captured target nucleic acids canthen be used in downstream analyses.

Thus, according to one embodiment of the present invention, there areprovided compositions and methods for the selective hybridization of aninactivatable target capture oligomer to a target nucleic acid, whereinsaid target nucleic acids is a DNA sequence or an RNA sequence; themethod comprises the steps of: (1) treating a target nucleic acidsequence in a nucleic acid sample with an inactivatable target captureoligomer in an active configuration, e.g., the tag-closing region andtarget hybridizing region are not stably hybridized one to the other,and under a first set of conditions for stably hybridizing the targethybridization region to a target nucleic acid sequence; and (2)inactivating any unhybridized inactivatable target capture oligomers byapplying a second set of conditions for stably hybridizing the targethybridization region and tag-closing region of the unhybridizedinactivatable target capture oligomer. The method can further comprisethe step of capturing said selectively hybridized target nucleic acid.Preferably, the capture step comprises using a solid support comprisinga second binding pair member complementary to the binding pair member ofthe inactivatable capture probe is introduced, thereby complexing withthe inactivatable target capture oligomer. Preferably, the first andsecond binding pair members are complementary and substantiallyhomopolymeric nucleotides sequences. Preferably, said solid support is amagnetic bead.

The present invention substantially reduces target capture of non-targetand contaminating nucleic acids. The term “non-target nucleic acid” isgenerally used herein to refer to nucleic acids, other than the targetnucleic acid, that are naturally present within the sample source,whereas the term “contaminating nucleic acids” is generally used hereinto refer to nucleic acids, other than the target nucleic acid, that areintroduced into a reaction mixture from a source external to the sample.Contaminating nucleic acids include, but are not limited to, nucleicacid that may be present in one or more reagents, components ormaterials that are used in an amplification reaction or that are presentin the environment in which an amplification reaction is performed aswell as nucleic acids from the sample source. The invention offers theadvantage of requiring less stringent reagent purification and/orsterility efforts than those conventionally used to ensure that enzymesand other reagents and components used in amplification reactions arefree of bacterial and other nucleic acid contaminants that may yieldfalse positive results. Such reagents or components include, but are notlimited to, water, buffers, salts, solid supports (e.g., magneticallycharged particles or beads), and receptacles (e.g., glassware orplasticware).

A mixture of nucleic acids may be those found in a sample, such as aclinical sample taken for diagnostic testing and screening of bloodproducts; sterility testing; seed stock testing; microbiologicaldetection from a food source, water source, beverage source, industrialsource or environmental source; research studies; whole cell lysates;reagent preparation and QC testing; or materials for other processessuch as cloning, or for other purposes where the presence of a targetnucleic acid may need to be detected and/or monitored. The selectiveamplification of specific nucleic acid sequences, as described herein,is of particular value in any of a variety of detection assays forincreasing the accuracy and reliability of such assays while at the sametime reducing the preparation, purification and/or sterilizationrequirements for reagents used in the assays. The compositions, methodsand reaction mixtures of the invention have particular advantages forthe testing raw materials used in the production of products for thebiotech, pharma, cosmetics and beverage industries, for release testingof final products, and for sterility screening to test for a class oforganisms or total viable organisms in a material of interest(bacterial, fungal or both). In the clinical setting, the methods andreaction mixtures of the invention would be particularly useful forsepsis testing, especially septicemia, which is caused by pathogenicorganisms and/or their toxins in the bloodstream.

Methods using the inactivatable target capture oligomers of the currentinvention can comprise an initial step of selectively hybridizing andcapturing a target nucleic acid followed by a subsequent nucleic acidanalysis of said target nucleic acid. By way of example only, a commonnucleic acid analysis is an amplification and detection assay. Oneexemplary amplification reaction comprises using an amplificationoligomer to introduce a tag sequence into the first amplificationproduct, and an additional amplification oligomer that hybridizes tosaid tag sequence (or complement thereof) to generate secondaryamplification products. Such is only an exemplary method, and any typeof downstream nucleic acid analysis, including amplification assays aswell as other types of assays, can be performed on selectivelyhybridized and captured target nucleic acids. For the exemplaryamplification reaction, following selective hybridization and capture ofa target nucleic acid under a first set of conditions and theninactivation of unhybridized inactivatable target capture oligomersunder a second set of conditions, a heterologous amplification oligomercomprising first and second regions is used in a first amplificationreaction to introduced a tag sequence into the target nucleic acidsequence. Thus, said first region of said heterologous amplificationoligomer comprises a target hybridizing sequence which hybridizes to a3′-end of the target nucleic acid sequence and said second regioncomprising a tag sequence situated 5′ to said target hybridizingsequence, and where said tag sequence does not stably hybridize to atarget nucleic acid containing the target nucleic acid sequence. Usingthis heterologous amplification oligomer in a first amplificationreaction, the first primer extension product introduces this tagsequence into the target nucleic acid sequence, providing a sequencethat is later targeted by a subsequent amplification oligomers. In onealternative aspect, this tag sequence is provided by incorporation ofthe tag-closing region of the inactivatable target capture oligomer.This aspect is accomplished, for example, by providing an inactivatabletarget capture oligomer that also serves as a primer for a firstsynthesis of a complementary strand of the target nucleic acid sequence.Here, the inactivatable target capture oligomer comprises a tagsequence, which can be either a forth region of the inactivatable targetcapture oligomer, can be a region that contains all or part of thetag-closing region or it can be the tag-closing sequence. Subsequentamplification then utilizes an amplification oligomer member thathybridizes with the tag region. Preferably, a wash step is includedfollowing hybridization of the inactivatable target capture oligomer or,in embodiments using a heterologous amplification oligomer comprising atag sequence, the wash step follows the hybridization of theinactivatable target capture oligomer and the heterologous amplificationoligomer. The wash step will remove inactivated and unhybridizedinactivatable target capture oligomers and other nucleic acid componentsin the reaction mixture. The first synthesis of a complementary strandthen takes place without the presence of these other nucleic acids inthe reaction mixture. Subsequently introduced contaminants are notamplified because these contaminants were neither captured nor do theyhave the tag sequence introduced into their sequences.

Embodiments wherein said target nucleic acid is modified to include atag sequence for subsequent hybridization by a heterologousamplification oligomer, said tag sequence can be introduced by using theinactivatable target capture oligomer for a primer-based reaction, thusincorporating the tag sequence into the amplification product, or,preferably, using a heterologous amplification oligomer in aprimer-based reaction to similarly incorporate the tag sequence into anamplification product. In a preferred embodiment, a heterologousamplification oligomer comprising a tag sequence and a targethybridizing sequence is hybridized to said target nucleic acid.Preferably, following hybridization of said heterologous amplificationoligomer to said target nucleic acid, the target nucleic acid, targetcapture oligomer and heterologous amplification oligomer complex iscaptured and the remaining components in the mix are washed away;including uncaptured and inactive inactivatable target capture oligomer,unhybridized heterologous amplification oligomer and non-target nucleicacids. A polymerase is then added to make a complementary target nucleicacid sequence from said heterologous amplification oligomer. Theresulting complementary target nucleic acid sequence has said tagsequence incorporated therein. The presence of non-target nucleic acidswas substantially reduced or eliminated in the wash step. The presenceof unhybridized heterologous amplification oligomer was substantiallyreduced or eliminated in the wash step. The presence of inactive andunhybridized inactivatable target capture oligomer was substantiallyreduced or eliminated in the wash step. Amplification of subsequentlyintroduced contaminant is thusly substantially reduced or eliminated,too, because of a lack of these reagents.

Preferably, following said step of making a complementary target nucleicacid sequence from said heterologous amplification oligomer, anamplification reaction is performed using at least two amplificationoligomers, one of which is complementary to said incorporated tagsequence. In this way, the amplification of subsequently introducedcontaminants is substantially reduced or eliminated because theamplification reaction is directed towards nucleic acids containing saidtag sequence. These subsequently used amplification oligomers includeprimer oligomers and promoter-based oligomers, such as a promoter primeror a promoter provider. Exemplary amplification reactions are asfollows:

A first amplification method comprises the steps of (a) treating atarget nucleic acid complex immobilized on a solid support, with aheterologous amplification oligomer comprising a tag sequence to producea heterologous amplification oligomer:target nucleic acid complex; (b)reducing in said sample the effective concentration of heterologousamplification oligomer sequences that have not formed part of saidcomplex; and (c) subjecting said target nucleic acid sequence toreagents and conditions sufficient for incorporation of said tagsequence into said target nucleic acid sequence and then for detectableamplification of the target nucleic acid sequence, where the subjectingstep exposes the sample to a known contaminating source of the targetnucleic acid sequence after step (b), and where detectable amplificationof the target nucleic acid sequence is substantially limited toamplification of target nucleic acid sequence contributed by the tagsequence of step (a) and not by the known contaminating source.

In one aspect of this embodiment, one or more reagents used in themethods, such as nucleic acid polymerases, are produced using amicroorganism containing the contaminating nucleic acid sequence. In afurther aspect, components used in the methods, such as reactionvessels, pipette tips and solid supports for binding complexescomprising the captured target nucleic acid sequences, may be a knowncontaminating source of the target nucleic acid sequence. In a furtheraspect, the methods are useful where the environmental conditions inwhich amplification is performed include a known contaminating source ofa nucleic acid sequence, such as the ambient air, operator or analyticalinstrumentation.

In a further aspect of this embodiment, the captured target nucleic acidsequence is immobilized on a solid support during step (b). Preferably,the target nucleic acid sequence and inactivatable target captureoligomer complex is immobilized on a solid support during step (b). Morepreferably, the target nucleic acid sequence, inactivatable targetcapture oligomer and heterologous amplification oligomer complex isimmobilized on a solid support during step (b).

In further aspect, step (b) comprises diluting or removing inactivatabletarget capture oligomers that have not hybridized to target nucleic acidsequences. Preferably, step (b) comprises diluting or removinginactivatable target capture oligomers and heterologous targetamplification oligomers that have not hybridized to target nucleic acidsequences.

In an alternative aspect, an inactivatable target capture oligomer maybe inactivated by blocking its ability to complex with a target nucleicacid sequence by using an enzyme to digest a component or cleave a siteof a portion of its nucleic acid sequence, chemically altering thesequence, or altering by other means the ability of the targethybridization sequence to complex with a nucleic acid sequence in areaction mixture.

In yet another aspect, the target hybridizing sequence of saidinactivatable target capture oligomer, in certain aspects, is auniversal oligonucleotide, such as a universal bacterial or fungaloligonucleotide. In yet another aspect, the target hybridizing sequenceof said heterologous amplification oligomer, in certain aspects, is auniversal oligonucleotide, such as a universal bacterial or fungaloligonucleotide. In yet another aspect, the target hybridizing sequenceof said inactivatable target capture oligomer and the target hybridizingsequence of said heterologous amplification oligomer are both universaloligonucleotide sequences, such as a universal bacterial or fungaloligonucleotide.

Step (c) comprises producing amplification products in a nucleic acidamplification reaction, wherein a complement of said target nucleic acidis made using the heterologous amplification oligomer thus resultingincorporating therein the tag sequence, and then using first and secondamplification oligonucleotides, wherein the first oligonucleotidecomprises a sequence which hybridizes to a 3′-end of the incorporatedtag sequence but which does not stably hybridize to the target nucleicacid sequence or the complement of the target nucleic acid sequence anda second oligonucleotide comprising, wherein each of the amplificationproducts comprises a base sequence which is substantially identical orcomplementary to the base sequence of the target nucleic acid sequenceand all or a portion of the tag sequence.

Various amplification methods are suitable for use in the presentinvention. For example, in one aspect, the amplification reaction is aPCR reaction. In another aspect, the target nucleic acid sequence isamplified by a transcription-based amplification reaction, preferably aTMA reaction, performed under isothermal conditions.

The target nucleic acid sequence amplified according to the methods canbe any target nucleic acid sequence of interest, but will generally be anucleic acid sequence obtained from a microorganism. Further, the methodcan be selective for the amplification of a target nucleic acid sequencecontained in the nucleic acid of a single strain or species ofmicroorganisms or in multiple species of microorganisms. Alternatively,the method can be selective for the amplification of multiple targetnucleic acid sequences contained in the nucleic acid of multiple speciesof microorganisms, where, for example, the target hybridizing sequenceof an inactivatable target capture oligomer hybridizes to a targetregion present in each of the multiple target nucleic acid sequences instep (a).

For example, in a particular aspect, the method is selective for theamplification of a target nucleic acid sequence contained in each of aplurality of target nucleic acids, and wherein the heterologous tagsequence produces a tagged target nucleic acid sequence with the targetnucleic acid sequence of each of the plurality of target nucleic acidspresent in the nucleic acid sample in step (a). In a more particularaspect, the target nucleic acid sequence contained in each of theplurality of target nucleic acids is the same nucleic acid sequence.

In another particular aspect, the method is selective for theamplification of multiple bacterial or fungal target nucleic acidsequences, e.g., wherein the multiple bacterial or fungal target nucleicacid sequences are ribosomal nucleic acid sequences. In anotherparticular aspect, the method is selective for the amplification oftarget nucleic acid sequences obtained from members of a group ofbacterial species. In another aspect, the method is selective for theamplification of target nucleic acid sequences obtained from members ofa group of fungal species. In another particular aspect, the method isselective for the microorganisms that would be detected in a sepsistest. In yet another aspect, at least a portion of a nucleic acid sampleused in the methods is obtained from a clinical, water, industrial,environmental, seed, beverage or food source. The methods areparticularly well suited, in certain aspects, for use in sterilitytesting or diagnostic testing for sepsis.

A further amplification method comprises the steps of: (a) treating anucleic acid sample comprising a target nucleic acid sequence with aninactivatable target capture oligomer and a heterologous amplificationoligomer under a set of conditions for selective hybridization of theinactivatable target capture oligomer and a heterologous amplificationoligomer to the target nucleic acid; (b) reducing in said nucleic acidsample the effective concentration of unhybridized inactivatable targetcapture oligomer having an active form in which a target hybridizingsequence of said unhybridized inactivatable target capture oligomer isavailable for hybridization to said a non-target and/or contaminatingnucleic acid sequence; and (c) producing amplification products in anucleic acid amplification reaction using the heterologous amplificationoligomer to incorporate a tag sequence into the complementary targetnucleic acid sequence then using first and second oligonucleotides toamplify the target nucleic acid, where the first oligonucleotidecomprises a hybridizing sequence which hybridizes to a 3′-end of thecomplement of the target nucleic acid sequence and the secondoligonucleotide comprises a hybridizing sequence which hybridizes to thecomplement of the tag sequence, but does not stably hybridize to thetarget nucleic acid, and where the amplification products comprises abase sequence which is substantially identical to or complementary tothe base sequence of the target nucleic acid sequence and all or aportion of the tag sequence.

In one aspect of the above methods, at least one target nucleic acidsequence is immobilized on a solid support during step (b). In anotheraspect, step (b) does not include the use of an enzyme having a nucleaseactivity.

The effective concentration of unhybridized inactivatable target captureoligomer in an active form prior to amplification is preferably reducedby diluting the nucleic acid sample or by inactivating and/or removingthe unhybridized inactivatable target capture oligomer. In one aspect,step (b) comprises inactivating unhybridized inactivatable targetcapture oligomer so that it does not stably hybridize to any nucleicacid sequence during step (c). In one example of inactivation, aninactivatable target capture oligomer has an active form during step (a)which permits the target hybridizing sequence to hybridize to the targetnucleic acid sequence, and where unhybridized inactivatable targetcapture oligomer is converted to an inactive form in step (b) whichblocks or prevents its hybridizing to the target nucleic acid sequenceduring step (c). In a related embodiment, the conditions of steps (b)and (c) are less stringent than the conditions of step (a). In anotherrelated embodiment, the temperature of the nucleic acid sample islowered between steps (a) and (b).

In another alternative example, unhybridized inactivatable targetcapture oligomer from step (a) is converted from a single-stranded formto a duplexed form in step (b). The duplexed form may be a hairpinmolecule comprising a tag-closing sequence, where the tag-closingsequence hybridizes to the target hybridizing sequence under theconditions of step (b), thereby blocking hybridization of unhybridizedinactivatable target capture oligomer from step (a) to the targetnucleic acid sequence in steps (b) and (c). In another aspect, thetag-closing sequence is joined to the inactivatable target captureoligomer by a non-nucleotide linker. In orientations of theinactivatable target capture oligomer wherein a terminal 3′ residue isavailable for primer based nucleic acid extension and such extension isnot desired, then the 3′ terminal residue can be blocked using ablocking moiety.

In still another aspect, the target hybridizing sequence is hybridizedto a tag-closing oligonucleotide in step (b), the inactivatable targetcapture oligonucleotide and the tag-closing oligonucleotide beingdistinct molecules. The tag-closing oligonucleotide may be modified, ifdesired, to prevent the initiation of DNA synthesis therefrom.

Further, in certain aspects, a 3′-terminal base of the targethybridizing sequence is hybridized to a 5′-terminal base of thetag-closing oligonucleotide.

As noted above, the methods of the invention can employ any of a varietyof amplification techniques. In certain instances it may be preferredthat an isothermal amplification reaction is used, such as atranscription-based amplification reaction, preferably TMA or real-timeTMA.

In a particular aspect, the first amplification oligonucleotidecomprises a promoter sequence for an RNA polymerase the promotersequence being situated 5′ to the target hybridizing sequence. Inanother aspect, the second oligonucleotide comprises a promoter sequencefor an RNA polymerase, the promoter sequence being situated 5′ to thetarget hybridizing sequence. In a further aspect, one or both of thefirst or second amplification oligomers comprises a target hybridizingregion, a tag sequence region and a promoter sequence region for an RNApolymerase, wherein the promoter sequence is situated 5′ to the tagsequence region.

The nucleic acid sample is often exposed to a known contaminating sourceafter step (b), and, accordingly, the described methods provide that theproduction of amplification products is substantially limited toamplification of target nucleic acid sequence contributed by the nucleicacid sample and not by the contaminating nucleic acid sequence. Forexample, one or more reagents or components used in the amplificationreaction comprise a known contaminating source. Alternatively, or inaddition, one or more reagents are produced with a material known to bea contaminating source; such as nucleic acid polymerases produced usingmicroorganisms known to contain the target nucleic acid sequence.Further, the environmental conditions in which the method is performedmay include a known contaminating source. In a particular aspect, atleast a portion of said nucleic acid is obtained from clinical, water,industrial, environmental, seed, beverage or food sources.

According to a third exemplary amplification method, the target nucleicacid sequence is an RNA target sequence, and step (c) comprises:extending the heterologous amplification oligomer hybridized to thetarget nucleic acid sequence in a primer extension reaction with a DNApolymerase to produce an extension product comprising a regioncomplementary to the target nucleic acid sequence and a tag sequence;separating the extension product from the target nucleic acid using anenzyme which selectively degrades that portion of the target nucleicacid hybridized to the extension product; treating the extension productwith a first amplification oligonucleotide, the first amplificationoligonucleotide being a promoter oligonucleotide comprising ahybridizing sequence which hybridizes to a region of the extensionproduct that is complementary to a 5′-end of the target nucleic acidsequence to form a promoter oligonucleotide: extension product hybrid,and comprising a promoter sequence for an RNA polymerase, the promotersequence being situated 5′ to the target hybridizing region;transcribing with an RNA polymerase using as a template the promoteroligonucleotide:extension product complex multiple copies of a first RNAproduct complementary to at least a portion of the extension product andincorporated tag sequence; treating the first RNA product with thesecond amplification oligonucleotide that hybridizes to the complementof the tag sequence to form a second amplification oligonucleotide:firstRNA product complex such that a primer extension reaction can beinitiated from the second amplification oligonucleotide; extending thesecond amplification oligonucleotide in a primer extension reaction witha DNA polymerase to produce a second primer extension productcomplementary to the first RNA product, the second primer extensionproduct having a 3′-end which is complementary to a 5′-end of the firstRNA product; separating the second primer extension product from thefirst RNA product using an enzyme which selectively degrades said firstRNA product; treating the second primer extension product with thepromoter oligonucleotide to form a promoter oligonucleotide:secondprimer extension product complex; extending a 3′-end of the secondprimer extension product in the promoter oligonucleotide:second primerextension product complex to add a sequence complementary to the secondregion of the promoter oligonucleotide; and transcribing from thepromoter oligonucleotide:second primer extension product hybrid multiplecopies of a second RNA product complementary to the second primerextension product using the RNA polymerase.

In another aspect of this embodiment of the invention, step (a) furthercomprises treating the nucleic acid sample with a binding molecule whichbinds to the target nucleic acid adjacent to or near a 5′-end of thetarget nucleic acid sequence, and where the first primer extensionproduct has a 3′-end which is determined by the binding molecule andwhich is complementary to the 5′-end of the target nucleic acidsequence.

In another aspect, step (c) of the above embodiment further comprisesextending a 3′-end of the first primer extension product in the promoteroligonucleotide:first primer extension product hybrid to add a sequencecomplementary to the promoter. In yet another aspect, the promoteroligonucleotide is modified to prevent the initiation of DNA synthesistherefrom.

The promoter oligonucleotide hybridized to the first primer extensionproduct is extended with a DNA polymerase to produce a primer extensionproduct complementary to the first primer extension product; and thepromoter oligonucleotide hybridized to said second primer extensionproduct is extended with a DNA polymerase to produce a primer extensionproduct complementary to the second primer extension product.

The separating steps of the described methods may be performed with aribonuclease activity provided by the DNA polymerase. Alternatively, theseparating steps are performed with a ribonuclease activity provided byan enzyme other than said DNA polymerase.

According to a forth amplification method, the target nucleic acidsequence is an RNA target sequence, and step (c) comprises: extendingthe heterologous amplification oligomer hybridized to the target nucleicacid sequence in a primer extension reaction with a DNA polymerase toproduce a first primer extension product comprising a regioncomplementary to the target nucleic acid sequence and a tag sequence,where the heterologous amplification oligonucleotide further comprises athird region situated 5′ to the tag sequence, the third regioncomprising a promoter for an RNA polymerase; separating the first primerextension product from the target nucleic acid using an enzyme whichselectively degrades that portion of the target nucleic acid hybridizedto the first primer extension product; treating the first primerextension product with a first amplification oligonucleotide, the firstamplification oligonucleotide being a priming oligonucleotide whichhybridizes to a region of the first primer extension product that iscomplementary to a 5′-end of the target nucleic acid sequence to form afirst amplification oligonucleotide:first primer extension productcomplex such that a primer extension reaction can be initiated from thefirst amplification oligonucleotide; extending the first amplificationoligonucleotide in a primer extension reaction with a DNA polymerase toproduce a second primer extension product complementary to the firstprimer extension product; and using the second primer extension productas a template to transcribe multiple copies of a first RNA productcomplementary to at least a portion of the second primer extensionproduct using an RNA polymerase which recognizes the promoter andinitiates transcription therefrom, where the base sequence of the firstRNA product is substantially identical to the base sequence of the tagsequence and the complement of the target nucleic acid sequence.

In another aspect of this embodiment, step (c) further comprises:treating the first RNA product with the second amplificationoligonucleotide to form a priming oligonucleotide: first RNA productcomplex such that a primer extension reaction can be initiated from thesecond amplification oligonucleotide; extending the primingoligonucleotide in a primer extension reaction with a DNA polymerase toproduce a third primer extension product complementary to the first RNAproduct, the third primer extension product having a 3′-end which iscomplementary to a 5′-end of the first RNA product; separating the thirdprimer extension product from the first RNA product using an enzymewhich selectively degrades the first RNA product; treating the thirdprimer extension product with the second oligonucleotide, the secondoligonucleotide being a promoter oligonucleotide comprising first andsecond regions, the first region comprising a hybridizing sequence whichhybridizes to the complement of the tag sequence to form a promoteroligonucleotide:third primer extension product hybrid such that a primerextension reaction can be initiated from the promoter oligonucleotide,and the second region comprising a promoter for an RNA polymerase whichis situated 5′ to the first region; extending the promoteroligonucleotide in a primer extension reaction with the DNA polymeraseto produce a fourth primer extension product complementary to the thirdprimer extension product; extending the third primer extension productto add a sequence complementary to the promoter; transcribing from thepromoter oligonucleotide:third primer extension product hybrid multiplecopies of a second RNA product complementary to the third primerextension product using an RNA polymerase which recognizes the promoterand initiates transcription therefrom, where the base sequence of thesecond RNA product is substantially identical to the base sequence ofthe tag sequence and the complement of the target nucleic acid sequence.

In another aspect of this embodiment, the separating steps are performedwith a ribonuclease activity provided by the DNA polymerase.Alternatively, the separating steps are performed with a ribonucleaseactivity provided by an enzyme other than the DNA polymerase.

According to fifth amplification method, the target nucleic acidsequence is a DNA target sequence, and step (c) comprises: extending theheterologous amplification oligomer hybridized to the target nucleicacid sequence in a primer extension reaction with a DNA polymerase toproduce a first primer extension product comprising a regioncomplementary to the target nucleic acid sequence a the tag sequence;treating the first primer extension product with the first amplificationoligonucleotide, the first amplification oligonucleotide being apromoter oligonucleotide comprising a target hybridizing sequence whichhybridizes to a region of the first primer extension product that iscomplementary to a 5′-end of the target nucleic acid sequence to form apromoter oligonucleotide:first primer extension product hybrid, and thesecond region being a promoter for an RNA polymerase which is situated5′ to the first region; transcribing from the promoteroligonucleotide:first primer extension product complex multiple copiesof a first RNA product complementary to at least a portion of the firstprimer extension product using an RNA polymerase that recognizes thepromoter and initiates transcription therefrom, where the base sequenceof the first RNA product is substantially identical to the base sequenceof the target nucleic acid sequence and the complement of the tagsequence; treating the first RNA product with the second amplificationoligonucleotide, the second amplification oligonucleotide being apriming oligonucleotide which hybridizes to the complement of the tagsequence to form a priming oligonucleotide:first RNA product hybrid suchthat a primer extension reaction can be initiated from the primingoligonucleotide; extending the priming oligonucleotide in a primerextension reaction with a DNA polymerase to give a second primerextension product comprising the complement of the first RNA product,the second primer extension product having a 3′-end which iscomplementary to a 5′-end of the first RNA product; separating thesecond primer extension product from the first RNA product using anenzyme which selectively degrades the first RNA product; treating thesecond primer extension product with the promoter oligonucleotide toform a promoter oligonucleotide:second primer extension product hybrid;extending a 3′-end of the second primer extension product in thepromoter oligonucleotide:second primer extension product hybrid to add asequence complementary to the promoter; and transcribing from thepromoter oligonucleotide:second primer extension product hybrid multiplecopies of a second RNA product complementary to the second primerextension product using the RNA polymerase, where the base sequence ofthe second RNA product is substantially identical to the base sequenceof the target nucleic acid sequence and the complement of the tagsequence.

In one aspect of this embodiment, the promoter oligonucleotide ismodified to prevent the initiation of DNA synthesis therefrom.

In another aspect, step (a) further comprises: treating the nucleic acidsample with a displacer oligonucleotide which hybridizes to the targetnucleic acid upstream from the heterologous amplificationoligonucleotide such that a primer extension reaction can be initiatedfrom the displacer oligonucleotide; and extending the displaceroligonucleotide in a primer extension reaction with a DNA polymerase toproduce a third primer extension product that displaces said firstprimer extension product from the target nucleic acid.

In yet another embodiment, step (a) further comprises treating thenucleic acid sample with a binding molecule which binds to the targetnucleic acid adjacent to or near a 5′-end of the target nucleic acidsequence, where the first primer extension product has a 3′-end which isdetermined by said binding molecule and which is complementary to the5′-end of the target nucleic acid sequence.

In a more particular aspect, step (c) further comprises extending a3′-end of the first primer extension product in the promoteroligonucleotide: first primer extension product complex to add asequence complementary to the promoter sequence.

In another particular aspect, step (c) further comprises: extending thepromoter oligonucleotide hybridized to the first primer extensionproduct with a DNA polymerase to produce a primer extension productcomplementary to the first primer extension product; and extending thepromoter oligonucleotide hybridized to the second primer extensionproduct with a DNA polymerase to produce a primer extension productcomplementary to the second primer extension product.

The separating steps, in one embodiment, are performed by a ribonucleaseactivity provided by said DNA polymerase. Alternatively, the separatingsteps are performed by a ribonuclease activity provided by an enzymeother than said DNA polymerase.

Another embodiment of the present invention provides a kit for use inthe selective hybridization of at least one inactivatable target captureoligomer to at least one target nucleic acid sequence from a nucleicacid sample, the kit comprising: an inactivatable target captureoligomer comprising a first region comprising a target hybridizingsequence which hybridizes to a target nucleic acid sequence under afirst set of conditions, a second region comprising a tag-closingsequence situated, preferably, 3′ to the first region, where the secondregion does not stably hybridize to a target nucleic acid under thefirst set of conditions and third region comprising a binding pairmember situated, preferably, 3′ to the second region. In one aspect,said kit further comprises a solid support comprising a second bindingpair member that is complementary to the binding pair member of theinactivatable target capture oligomer. In a further aspect, the solidsupport is a magnetic bead. In a further aspect, the first binding pairmember of the inactivatable target capture oligomer and the secondbinding pair member of the solid support are complementary nucleic acidsequences, preferably said sequences are substantially homopolymeric.

In a further embodiment, said kit comprises a heterologous amplificationoligomer comprising a target hybridizing region that hybridizes totarget nucleic acid sequence and a tag sequence that doe not stablyhybridize to the target nucleic acid sequence. In a further embodiment,the kit comprises at least two amplification oligomers, one of which ishybridizes to the tag sequence, or complement thereof. Amplificationoligomers include primers, promoter-based amplification oligomer andheterologous amplification oligomers. In a further embodiment, the kitcomprises a terminating oligomer.

In yet another aspect, the tag-closing sequence includes a moiety forstabilizing a duplex formed between the tag-closing sequence and thetarget hybridizing sequence when the target hybridizing sequence is nothybridized to the target nucleic acid sequence under the second set ofconditions.

In another and alternative aspect, the inactivatable target captureoligonucleotide comprises a tag-closing sequence that constitutes adistinct molecule from the molecule comprising the target hybridizingsequence and the binding pair member.

The tag-closing sequence may be joined to the target hybridizing regionby a non-nucleotide linker, for example a non-nucleotide linkercomprising at least one of abasic nucleotides and polyethylene glycol.In another aspect, a 3′-end of the tag-closing sequence is joined to a5′-end of the target hybridizing region. Alternatively, a 5′-end of thetag-closing sequence is joined to a 5′-end of the target hybridizingregion. Alternatively, the tag-closing region and the target hybridizingregion are joined in a 5′- to ‘5’ or 3′ to ‘3’ orientation using anon-nucleotide linker. In yet another aspect, the tag-closing sequencehybridizes to the target hybridizing sequence to form an antiparallelduplex when the target hybridizing sequence is not hybridized to thetarget nucleic acid sequence under the second set of conditions. Inother aspects wherein the orientation and/or arrangement of the regionsof the inactivatable target capture oligomer provides a terminal 3′ endthat can be extended by a polymerase in a priming event and wherein suchextension is not desired, the 3′ terminal nucleotide of a sequence ismodified to prevent the initiation of DNA synthesis therefrom, forexample by including a blocking moiety situated at its 3′-terminus.

In another aspect, the tag-closing sequence hybridizes to the targethybridizing sequence to form a parallel duplex when the targethybridizing sequence is not hybridized to the target nucleic acidsequence under the second set of conditions.

In yet another aspect, the duplex comprises a 3′-terminal base of thetarget hybridizing sequence hybridized to a 3′-terminal base of thetag-closing sequence. In another aspect of this embodiment, the firstpriming oligonucleotide does stably hybridize to the target nucleic acidand, thereby, participates in detectable amplification of the targetnucleic acid sequence under the second set of conditions. In anotheraspect, the kit further comprises a second priming oligonucleotide whichhybridizes to the complement of a 5′-end of the target nucleic acidsequence under the second set of conditions so that the second primingoligonucleotide can be extended in a template-dependent manner in thepresence of a DNA polymerase. In yet another aspect, a kit of theinvention further comprises a promoter oligonucleotide comprising firstand second regions, the first region comprising a hybridizing sequencewhich hybridizes to the complement of a 5′-end of the target nucleicacid sequence under the second set of conditions, and the second regioncomprising a promoter for an RNA polymerase which is situated 5′ to thefirst region. The promoter oligonucleotide, in this aspect, may bemodified to prevent the initiation of DNA synthesis therefrom, forexample by including a blocking moiety situated at its 3-terminus. Inyet another aspect, the promoter oligonucleotide can be extended in atemplate-dependent manner in the presence of a DNA polymerase when thehybridizing sequence is hybridized to the complement of the 5′-end ofthe target nucleic acid sequence under the second set of conditions.

The kits of the invention, in certain aspects, may also further compriseone or more reagents or components selected from any one or more of aDNA polymerase (such as a reverse transcriptase), an RNA polymerase,nucleoside triphosphates, a solid support for binding a complexcomprising the target nucleic acid and the inactivatable target captureoligonucleotide. In another aspect, the inactivatable target capture isfree in solution. In another aspect, the kit does not include arestriction enzyme capable of cleaving a duplex formed between thetag-closing sequence and the target hybridizing sequence under thesecond set of conditions. In yet another aspect, the target hybridizingsequence hybridizes to a 3′-end of multiple target nucleic acidsequences under the first set of conditions.

According to another embodiment of the invention, there is provided apreannealing reaction mixture for stably hybridizing an activeinactivatable target capture oligomer to target nucleic acid sequenceunder a first set of conditions. In another embodiment, there isprovided a preannealing reaction mixture for stably hybridizing anactive inactivatable target capture oligomer and a heterologousamplification oligomer to target nucleic acid sequence under a first setof conditions. In another embodiment there is provided a preannealingreaction mixture for stably hybridizing an active inactivatable targetcapture oligomer and, optionally, a heterologous amplification oligomerto target nucleic acid sequence under a first set of conditions, and forinactivating unhybridized and active inactivatable target captureoligomer under a second set of conditions. In another embodiment thereis provided a preannealing reaction mixture for stably hybridizing anactive inactivatable target capture oligomer and a heterologousamplification oligomer to target nucleic acid sequence under a first setof conditions, for inactivating unhybridized and active inactivatabletarget capture oligomer under a second set of conditions, and forreducing or eliminating non-target nucleic acid, inactivated targetcapture oligomer and heterologous amplification oligomer from thereaction mixture. In another embodiment there is provided a preannealingreaction mixture for stably hybridizing an active inactivatable targetcapture oligomer and a heterologous amplification oligomer to targetnucleic acid sequence under a first set of conditions, for inactivatingunhybridized and active inactivatable target capture oligomer under asecond set of conditions, and for reducing or eliminating non-targetnucleic acid, inactivated target capture oligomer and heterologousamplification oligomer from the reaction mixture, wherein said targetnucleic acid is subsequently used in a nucleic acid analysis assay. Inanother embodiment there is provided a preannealing reaction mixture forstably hybridizing an active inactivatable target capture oligomer and aheterologous amplification oligomer to a target nucleic acid sequenceunder a first set of conditions, for inactivating unhybridized andactive inactivatable target capture oligomer under a second set ofconditions, and for reducing or eliminating non-target nucleic acid,inactivated target capture oligomer and heterologous amplificationoligomer from the reaction mixture, wherein said heterologousamplification oligomer is used to make a complementary copy of thetarget nucleic acid containing the tag sequence.

In yet another aspect, the inactivatable target capture oligonucleotideis not attached to a solid support.

Certain other embodiments of the invention relate to the use of themethods described herein as a means for monitoring bioprocess samples,streams, and the like. In one embodiment, for example, there is provideda method for monitoring a bioprocess for the presence of contaminatingnucleic acid comprising the steps of (a) treating a first bioprocesssample with inactivatable target capture oligomer and, optionally, aheterologous amplification oligomer under conditions wherein theinactivatable target capture oligomer and the heterologous amplificationoligomer, if present, stably hybridize to the target nucleic acidsequence present in said first sample; (b) removing or inactivatingunhybridized inactivatable target capture oligomer from the firstbioprocess sample; and (c) exposing a second bioprocess sample, thesecond bioprocess sample comprising the first bioprocess sample andfurther comprising additional bioprocess samples, to amplificationreagents and conditions sufficient for amplification of the targetnucleic acid sequence using an amplification reaction generally asdescribed or referenced herein.

In another embodiment, the present invention provides a method formonitoring a bioprocess for the presence of contaminating nucleic acidcomprising the steps of (a) treating a first bioprocess sample with afirst inactivatable target capture oligomer and, optionally, a firstheterologous amplification oligomer under conditions where the firstinactivatable target capture oligomer and the first heterologousamplification oligomer, if present, stably hybridize to the targetnucleic acid sequence present in said first sample; (b) treating asecond bioprocess sample with a second inactivatable target captureoligomer and, optionally, a second heterologous amplification oligomer;under conditions where the second inactivatable target capture oligomerand the second heterologous amplification oligomer, if present, stablyhybridize to the target nucleic acid sequence present in the secondsample; and (c) performing a nucleic acid amplification reaction on athird bioprocess sample, the third bioprocess sample comprising thefirst and the second bioprocess samples, using: (i) a firstamplification oligonucleotide which hybridizes to a complement of thefirst tag sequence; (ii) a second amplification oligonucleotide sequencewhich hybridizes to a complement of the second tag sequence; and (iii) athird amplification oligonucleotide which hybridizes to a complement ofthe target nucleic acid sequence; where the detection of amplificationproduct resulting from the first and third amplificationoligonucleotides is indicative of the presence of the target nucleicacid sequence of the organism of interest in the first bioprocesssample, and where detection of amplification product resulting from thesecond and third oligonucleotides is indicative of the presence of thetarget nucleic acid sequence of the organism of interest in the secondbioprocess sample.

In a further embodiment of the invention, a pre-amplification reactionmixture is provided for the selective amplification of one or moretarget nucleic acid sequences, where the reaction mixture comprises: aninactivatable target capture oligomer hybridized to a target nucleicacid and a heterologous amplification oligomer hybridized to a targetregion contained at a 3′-end of one or more target nucleic acidsequences present in the reaction mixture; a first amplificationoligonucleotide comprising a target hybridizing sequence whichhybridizes to a 3′-end of the complement of one or more of the targetnucleic acid sequences; and a second amplification oligonucleotidecomprising a hybridizing sequence which hybridizes to the complement ofthe tag sequence, where the second amplification oligonucleotidepreferably does not stably hybridize to a target nucleic acid, where thereaction mixture is substantially free of an active form of theinactivatable target capture oligomer, and where the reaction mixturedoes not include a nucleic acid polymerase capable of extending any ofthe oligonucleotides in a template-dependent manner. The “non-target”nucleic acid is from a source outside of the reaction mixture and maycontain a sequence identical to that of the target nucleic acidsequence. The source of the non-target nucleic acid may be environmentalor it may be a component or reagent added to the reaction mixture, sucha nucleic acid polymerase. Each one of the heterologous amplificationoligomer, first amplification oligomer or second amplification oligomercan be a priming oligonucleotide or a promoter-based oligonucleotide.

In one embodiment, inactivatable target capture oligomers that have nothybridized to the target region of at least one target nucleic acidsequence remain “free” in the reaction mixture (i.e., the inactivatabletarget capture oligomers do not form hybrid duplexes other than throughself-hybridization). Self-hybridized inactivatable target captureoligomers are referred to as “hairpin tag molecules,” which are theinactive forms of the molecule that prevents it from hybridizing to anycomplementary nucleic acids that are subsequently added to the reactionmixture, such as through a contaminated enzyme preparation or reagentcontaining non-target nucleic acids. In still another aspect of thisembodiment, substantially all of the inactivatable target captureoligomers in the reaction mixture are in a hybridized state (hybridizedeither to the target region of a target nucleic acid sequence or tothemselves in the form of hairpin tag molecules). At least a portion ofthe tag molecules which have not hybridized to the target region of atarget nucleic acid sequence (i.e., hairpin tag molecules) are removedfrom the reaction mixture by, for example, subjecting the reactionmixture to a target capture and washing procedure.

In a still further aspect of this embodiment, there are substantially noinactivatable target capture oligomers that exist in an unhybridizedstate when the reaction mixture is exposed to an enzyme preparation foramplifying the one or more target nucleic acid sequences. Thus, in thisaspect, the reaction mixture is substantially depleted of unhybridizedinactivatable target capture oligomers specific for the one or moretarget nucleic acid sequences provided by the sample of interest. Morepreferably, the reaction mixture is further substantially depleted ofself-hybridized (inactivated) inactivatable target captureoligonucleotides. This may be accomplished with, for example, a targetcapture, reaction mixture conditions and washing procedure that separatehybridized inactivatable target capture oligonucleotides fromunhybridized inactivatable target capture oligonucleotides, and thenselectively removes the unhybridized inactivatable target captureoligonucleotides from the reaction mixture.

In some embodiments a probe-based detection assay is performed fordetecting target nucleic acid sequences. Thus in one aspect, a probe isincluded for detecting an amplification product synthesized in an invitro reaction that involves enzymatic synthesis of a target nucleicacid sequence. In other aspects, a target nucleic acid sequence iscaptured using the compositions and methods of the current invention andthe captures target nucleic acid is detected by direct hybridization ofa probe molecule. Amplification products include nucleic acid copies ofone or more of the target nucleic acid sequences and/or theircomplements.

In yet another embodiment, a reaction mixture is provided for theselective amplification of one or more target nucleic acid sequences,where the reaction mixture comprises: inactivatable target captureoligomer; a heterologous amplification oligomer; a first amplificationoligonucleotide comprising a hybridizing sequence which hybridizes to a3′-end of the complement of the target nucleic acid sequence; and asecond amplification oligonucleotide comprising a hybridizing sequencewhich hybridizes to the complement of the tag sequence, where the secondamplification oligonucleotide preferably does not stably hybridize to atarget nucleic acid containing the target nucleic acid sequence, andwhere substantially all unhybridized inactivatable target captureoligomer in the reaction mixture has an inactive form which blocks orprevents said unhybridized inactivatable target capture from hybridizingto the target nucleic acid sequence.

The inactive form of the inactivatable target capture oligomer cancomprise a tag-closing sequence hybridized to the target hybridizingsequence. The tag-closing sequence can be a distinct molecule when nothybridized to the target hybridizing sequence or it can be an integralpart of the inactivatable target capture oligomer sequence. An integraltag-closing sequence can be a continuous nucleotide sequence along withthe target hybridizing region or it can be joined to the targethybridizing region using a non-nucleotide linker (i.e., the constituentsof the linker cannot be copied by a nucleic acid polymerase). Theinactivatable target capture oligomer may or may not be joined to asolid support and is preferably not directly attached to solid support(e.g., particles or beads). If joined to a solid support, eitherdirectly or indirectly, the inactivatable target capture oligomer mayfurther function as a capture probe for binding and immobilizing atarget nucleic acid sequence.

The inactivatable target capture oligomer of the above reaction mixtureembodiments may possess the characterizing features of any of thevarious inactivatable target capture oligomer embodiments describedinfra. And, unless specifically excluded, the reaction mixtures mayfurther include the reagents and components needed to conduct anamplification reaction.

These and other features and advantages of the present invention willbecome apparent upon reference to the following detailed description,the attached drawings and the claims. All references disclosed hereinare hereby incorporated by reference in their entirety as if each wasincorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 through 16 illustrate exemplary amplification analysis reactionsthat can be performed using target nucleic acids that have been capturedusing the inactivatable target capture oligomers of the currentinvention. FIGS. 17 through 21 illustrate results from downstreamanalysis of selectively captured target nucleic acids, exemplifying thatthe compositions and methods of the current invention provide selectivehybridization and capture of target nucleic acids from samples. FIGS. 22through 25 illustrate some configurations of the inactivatable targetcapture oligomer of the current invention, in representative active andin inactive configurations. FIGS. 26 and 27 illustrate some method stepsof the selective capture and capture of a target nucleic acid sequenceusing the compositions of the current invention. Also illustrated inthese figures is the optional preannealing of an amplification oligomerand blocker oligomer for a preamplification step. This optional part ofthe illustration is not limiting, but rather is exemplary. The type ofamplification oligomer used in this optional step can be a heterologousamplification oligomer, a primer oligomer or a promoter oligomer, asdescribed herein.

FIG. 1 illustrates the steps of a transcription-based amplificationreaction initiated with a tagged priming oligonucleotide (e.g., aheterologous amplification oligomer) that hybridizes to a 3′-end of anRNA target sequence. A first extension product formed with the taggedpriming oligonucleotide has a 3′-end which is determined by aterminating oligonucleotide hybridized adjacent to or near the 5′-end ofthe RNA target sequence. A blocked promoter oligonucleotide hybridizesto a 3′-end of the first extension product and is used to generate RNAtranscripts that are cycled into the amplification reaction.

FIG. 2 illustrates the use of a tag-closing sequence to form a hairpintagged priming oligomer molecule in the amplification reaction of FIG.1.

FIGS. 3A and 3B illustrate the steps of a transcription-mediatedamplification reaction initiated with a heterologous amplificationoligomer that is a tagged promoter oligonucleotide that hybridizes to a3′-end of an RNA target sequence.

FIG. 4 illustrates the use of a tag-closing sequence to form a hairpintagged promoter oligonucleotide in the amplification reaction of FIGS.3A and 3B.

FIG. 5 illustrates the steps of a transcription-based amplificationreaction initiated with a heterologous amplification oligomer that is atagged priming oligonucleotide that hybridizes to a 3′-end of asingle-stranded DNA target sequence. A first extension product formedwith the tagged priming oligonucleotide has a 3′-end which is determinedby a terminating oligonucleotide hybridized adjacent to or near the5′-end of the DNA target sequence. A displacer oligonucleotidehybridized 5′ to the tagged priming oligonucleotide is extended to forma second extension product which displaces the first extension productfrom the DNA target sequence. A blocked promoter oligonucleotidehybridizes to a 3′-end of the first extension product and is used togenerate RNA transcripts that are cycled into the amplificationreaction.

FIG. 6 illustrates the use of a tag-closing sequence to form a hairpintagged priming molecule in the amplification reaction of FIG. 5.

FIG. 7 illustrates the steps of a polymerase chain reaction that isinitiated with a heterologous amplification oligomer that is a taggedpriming oligonucleotide that hybridizes to a DNA target sequence.

FIG. 8 illustrates the use of a tag-closing sequence to form a hairpintagged priming molecule in the amplification reaction of FIG. 7.

FIG. 9 illustrates the steps of a reverse transcription polymerase chainreaction initiated with a heterologous amplification oligomer that is atagged priming oligonucleotide that hybridizes to an RNA targetsequence.

FIG. 10 illustrates the use of a tag-closing sequence to form a hairpintagged priming molecule in the amplification reaction of FIG. 9.

FIG. 11 illustrates a discrete, 3′ blocked tag-closing sequencehybridized in an antiparallel fashion to the 3′-end of a tagged primingoligonucleotide, thereby blocking hybridization of the tagged primingoligonucleotide to a target nucleic acid sequence.

FIG. 12 illustrates a discrete, 3′ blocked tag-closing sequencehybridized in an antiparallel fashion to the 3′-end of a tagged promoteroligonucleotide, thereby blocking hybridization of the tagged promoteroligonucleotide to a target nucleic acid sequence.

FIG. 13 illustrates a hairpin tag molecule that includes a 3′ blockedtag-closing sequence hybridized in a parallel fashion to the 3′-end of aheterologous amplification oligomer that is a tagged primingoligonucleotide, thereby blocking hybridization of the tagged primingoligonucleotide to a target nucleic acid sequence. The 5′-end of thetag-closing sequence is joined by a non-nucleotide linker to the 5′-endof a tag sequence of the tagged priming oligonucleotide.

FIG. 14 illustrates a hairpin tag molecule that includes a 3′ blockedtag-closing sequence hybridized in a parallel fashion to the 3′-end of aheterologous amplification oligomer that is a tagged promoteroligonucleotide, thereby blocking hybridization of the tagged promoteroligonucleotide to a target nucleic acid sequence. The 5′-end of thetag-closing sequence is joined by a non-nucleotide linker to the 5′-endof a promoter sequence of the tagged promoter oligonucleotide.

FIG. 15 illustrates a hairpin tag molecule that includes a 3′ blockedtag-closing sequence hybridized in an antiparallel fashion to the 3′-endof a heterologous amplification oligomer that is a tagged primingoligonucleotide, thereby blocking hybridization of the tagged primingoligonucleotide to a target nucleic acid sequence. The 5′-end of thetag-closing sequence is joined by a non-nucleotide linker to the 5′-endof a tag sequence of the tagged priming oligonucleotide.

FIG. 16 illustrates a hairpin tag molecule that includes a 3′ blockedtag-closing sequence hybridized in an antiparallel fashion to the 3′-endof a heterologous amplification oligomer that is a tagged promoteroligonucleotide, thereby blocking hybridization of the tagged promoteroligonucleotide to a target nucleic acid sequence. The 5′-end of thetag-closing sequence is joined by a non-nucleotide linker to the 5′-endof a promoter sequence of the tagged promoter oligonucleotide.

FIG. 17 shows the raw curves for HCV amplifications in which no targetwas spiked into the amplification reagent. There was no detectableamplification when the HCV transcript was not spiked into the targetcapture or amplification reagents, while the average TTime for reactionscontaining 1×10.sup.6 copies of the HCV transcript in the target capturereagent was 6.3 minutes.

FIG. 18 shows the raw curves for HCV amplifications in which target wasspiked into the amplification reagent. There was no detectableamplification when the HCV transcript was spiked into the amplificationreagent, while the average TTime for reactions containing 1×10.sup.6copies of the HCV transcript in the target capture reagent was 6.3minutes. The zero samples in target capture did not amplify, even with 1million copies HCV 1a spiked into the amplification reagent.

FIG. 19 shows the raw curves for HCV amplifications in which target andtagged nonT7 primer were spiked into the amplification reagent. TheAverage TTime for 1 million copies HCV 1a target present only in thetarget capture step with tagged nonT7 primer & terminatingoligonucleotide spiked into the amplification reagent was 7.2 minutes.The zero samples in target capture with target, terminatingoligonucleotide & tagged nonT7 primer spiked into the amplificationreagent also produced robust amplification with an Average TTime=8.6minutes.

FIG. 20 is graph that shows the results from time-dependent monitoringof nucleic acid amplification reactions that included either 0 or10.sup.6 copies of a synthetic E. coli rRNA template. The thin brokenline shows the results for the reaction conducted using 0 copies oftemplate. The heavy solid line shows the results for the reactionconducted using 10.sup.6 copies of template.

FIG. 21 is graph that shows the results from time-dependent monitoringof nucleic acid amplification reactions that included 0, 10.sup.3 or10.sup.5 copies of a synthetic E. coli rRNA template.

FIG. 22 is an exemplary illustration of inactivatable target captureoligomers wherein the tag-closing sequence is a separate molecule. As isdiscussed herein, inactivatable target capture oligomers can comprisetail regions and target hybridizing regions in the 5′ to 3′ orientationor in the 3′ to 5′ orientation. In an orientation wherein a targethybridizing region comprises a terminal 3′ residue, then that residuecould be used for primer based extension by a polymerase. If suchextension is not desired, then the 3′ residue can be blocked. Theorientation of the separate tag-closing sequence is determined by theorientation of the target hybridizing region.

FIG. 23 is an exemplary illustration of inactivatable target captureoligomers wherein the tag-closing sequence is joined to the targethybridization region by a non-nucleotide linker. As is discussed herein,inactivatable target capture oligomers can comprise tail regions andtarget hybridizing regions in the 5′ to 3′ orientation or in the 3′ to5′ orientation. In an orientation wherein a target hybridizing regioncomprises a terminal 3′ residue, then that residue could be used forprimer based extension by a polymerase. If such extension is notdesired, then the 3′ residue can be blocked.

FIGS. 24 and 25 are two exemplary illustrations of inactivatable targetcapture oligomers wherein the tag-closing sequence is an integralsequence of the target capture oligomer. In FIG. 24, the binding pairmember region is joined to the tag-closing region via a non-nucleotidelinker. As is discussed herein, inactivatable target capture oligomerscan comprise target hybridizing, tag-closing, and binding pair member,if a nucleic acid sequence, that are in either of the 5′ to 3′orientation or the 3′ to 5′ orientation. In an orientation wherein atarget hybridizing region comprises a terminal 3′ residue, then thatresidue could be used for primer based extension by a polymerase. Ifsuch extension is not desired, then the 3′ residue can be blocked.

FIG. 26 illustrates the selective hybridization of an inactivatabletarget capture oligomer to a target nucleic acid sequence. Selectivelyhybridized target nucleic acid sequences can be used for any of a numberof downstream purposes, as discussed herein. In this illustration thedownstream purpose is an amplification reaction. So, the target nucleicacid is optionally hybridized with an amplification oligomer and ablocker. The number of oligomers and target in a reaction will notnecessarily be 1:1, thus at each of parts c and d, the point is madethat following selective hybridization one or more of the target ofnucleic acid reagents can be in excess; denoted by m-o and n-o whereinm, n and o are numbers. The active form of the inactivatable targetcapture oligomer is illustrated in parts b and c. The hybridized form onthe inactivatable capture probe is shown in parts c and d. The inactiveform of the inactivatable target capture oligomer is illustrated in partd. The primer and terminating oligomer present in the figure are notrequired for the selective hybridization and capture of a target nucleicacid. These optional oligomers are illustrated merely for showing theembodiments wherein captures target nucleic acid is subsequentlyanalyzed in an amplification assay.

FIG. 27 illustrates capture of the selectively hybridized target nucleicacid. Following capture, the excess target and/or nucleic acid reagentsare removed by a wash. The selectively captured target nucleic acid canthen be used in a variety of downstream application, as described hereinor as known in the art. In the FIG. 27, the selectively captured targetnucleic acid is illustrated as being useful for a downstreamamplification reaction in that a primer is additionally hybridized tothe target. The primer and terminating oligomer present in the figureare not required for the selective hybridization and capture of a targetnucleic acid. These optional oligomers are illustrated merely forshowing the embodiments wherein captures target nucleic acid issubsequently analyzed in an amplification assay.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, compositions, kits and methodsare provided for the selective hybridization and capture of a targetnucleic acid, wherein these compositions, kits and methods desirablyreduce or eliminate the hybridization and capture of non-target nucleicacids and/or contaminating nucleic acids. Selectively hybridized andcaptured target nucleic acids are then used in any of a variety ofdownstream applications. One such downstream application is a nucleicacid amplification and detection assay. Using the compositions andmethods for selective target hybridization and capture of a targetnucleic acid followed by an amplification reaction, the false positiveamplification signals resulting from contaminating biological materialthat may be present in a reagent or component of an amplificationreaction are substantially reduced or eliminated compared to assays thatdo not use the invention compositions and methods. The providedcompositions and methods also allow for less stringent purificationand/or sterility efforts than have been conventionally needed in orderto ensure that enzymes and other reagents or components used inamplification reactions, and the environment in which amplificationreactions or other analyses are performed, are free of contamination bymicroorganisms or components thereof, that may yield false positiveresults.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, recombinantDNA, and chemistry, which are within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., MolecularCloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold SpringHarbor Laboratory Press: (1989); DNA Cloning, Volumes I and II (D. N.Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al., U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B.D. Hames & S. J. Higgins eds. 1984); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the treatise, Methods In Enzymology (AcademicPress, Inc., N.Y.); and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).

Definitions

The following terms have the following meanings unless expressly statedto the contrary. It is to be noted that the term “a” or “an” entityrefers to one or more of that entity; for example, “a nucleic acid,” isunderstood to represent one or more nucleic acids. As such, the terms“a” (or “an”), “one or more,” and “at least one” can be usedinterchangeably herein.

The term “nucleic acid” is intended to encompass a singular “nucleicacid” as well as plural “nucleic acids,” and refers to any chain of twoor more nucleotides, nucleosides, or nucleobases (e.g.,deoxyribonucleotides or ribonucleotides) covalently bonded together.Nucleic acids include, but are not limited to, viral genomes, orportions thereof, either DNA or RNA, bacterial genomes, or portionsthereof, fungal, plant or animal genomes, or portions thereof, messengerRNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), plasmid DNA,mitochondrial DNA, or synthetic DNA or RNA. A nucleic acid may beprovided in a linear (e.g., mRNA), circular (e.g., plasmid), or branchedform, as well as a double-stranded or single-stranded form. Nucleicacids may include modified bases to alter the function or behavior ofthe nucleic acid, e.g., addition of a 3′-terminal dideoxynucleotide toblock additional nucleotides from being added to the nucleic acid. Asused herein, a “sequence” of a nucleic acid refers to the sequence ofbases that make up a nucleic acid. The term “polynucleotide” may be usedherein to denote a nucleic acid chain. Throughout this application,nucleic acids are designated as having a 5′-terminus and a 3′-terminus.Standard nucleic acids, e.g., DNA and RNA, are typically synthesized“3′-to-5′,” i.e., by the addition of nucleotides to the 5′-terminus of agrowing nucleic acid.

A “nucleotide” is a subunit of a nucleic acid consisting of a phosphategroup, a 5-carbon sugar and a nitrogenous base. The 5-carbon sugar foundin RNA is ribose. In DNA, the 5-carbon sugar is 2′-deoxyribose. The termalso includes analogs of such subunits, such as a methoxy group at the2′ position of the ribose (2′-O-Me). As used herein, methoxyoligonucleotides containing “T” residues have a methoxy group at the 2′position of the ribose moiety, and a uracil at the base position of thenucleotide.

A “non-nucleotide unit” is a unit that does not significantlyparticipate in hybridization of a polymer. Such units must not, forexample, participate in any significant hydrogen bonding with anucleotide, and would exclude units having as a component one of thefive nucleotide bases or analogs thereof.

A “target nucleic acid” is a nucleic acid present in a nucleic acidsample comprising a “target sequence” to be selectively hybridized by aninactivatable target capture oligomer of the current invention. Targetnucleic acids may be DNA or RNA as described herein, and may be eithersingle-stranded or double-stranded. The target nucleic acid may includeother sequences besides the target sequence. Typical target nucleicacids include viral genomes, bacterial genomes, fungal genomes, plantgenomes, animal genomes, rRNA, tRNA, or mRNA from viruses, bacteria oreukaryotic cells, mitochondrial DNA, or chromosomal DNA.

Target nucleic acids may be from any number of sources. Sources oftarget nucleic acids include, but are not limited to, clinical specimens(e.g., blood, either whole blood or platelets, urine, saliva, feces,semen, or spinal fluid), environmental samples (e.g., water or soilsamples), food samples, beverages, industrial samples (e.g., productsand process materials, including water), seed stocks, cDNA libraries,whole cell lysates or total cellular RNA. By “isolated” it is meant thata sample containing a target nucleic acid is taken from its naturalmilieu; however, the term does not connote any particular degree ofpurification. If necessary, target nucleic acids of the presentinvention are made available for interaction with the variousoligonucleotides of the present invention, using methodologies such as,cell lysis or cell permeabilization to release the target nucleic acidfrom cells. Nucleic acids comprising a mixture of target nucleic acidsand non-target nucleic acids are then released. See, e.g., Clark et al.,U.S. Pat. No. 5,786,208; and Hogan, U.S. Pat. No. 6,821,770.

The term “target sequence” refers to the particular nucleotide sequenceof the target nucleic acid that is to be hybridized by a targethybridizing region. Further, “target sequence” can also includeparticular nucleotide sequence of the target nucleic acid that is to beamplified in a subsequent amplification reaction. Where the targetnucleic acid is originally single-stranded, the term “target sequence”will also refer to the sequence complementary to the “target sequence”as present in the target nucleic acid. Where the “target nucleic acid”is originally double-stranded, the term “target sequence” refers to boththe sense (+) and antisense (−) strands. In choosing a target sequence,the skilled artisan will understand that a sequence should be chosen soas to distinguish between unrelated or closely related target nucleicacids. As will be understood by those of ordinary skill in the art,these sequences are judged from the testing environment. At least thesequences recognized by the target hybridizing sequence of aninactivatable target capture oligomer should be dissimilar to othersequences in the environment being tested, but need not be unique withinthe universe of all possible sequences. Furthermore, it is not alwaysthe case that the unique sequence is in a single type of target nucleicacid. What this means is that in some embodiments, it may be desirableto choose a target sequence that is common to a class of organisms, forexample, a target nucleic acid sequence common to all E. coli strainsthat might be in a sample. In other situations, a very highly specifictarget sequence, or a target sequence having at least a highly specificregion recognized by the detection probe, would be chosen so as todistinguish between closely related organisms, for example, betweenpathogenic and non-pathogenic E. coli. In these instances, the targetsequence for these plurality of target nucleic acids should be uniqueamongst other sequences in the mixture, but are sufficiently identicalamongst themselves to stably hybridize to a common target hybridizingregion. A target sequence of the present invention may be of anypractical length. A minimal target sequence includes a region thathybridizes to a target hybridizing sequence and the complement thereof.Other considerations for determining length of a target sequence are,for example, the amount of secondary structure, or self-hybridizingregions in the sequence. Those of ordinary skill in the art usingroutine optimization methods easily accomplish determining the optimallength. Typically, target sequences of the present invention range fromabout 10 nucleotides in length, to about 100 nucleotides in length tofrom about 150 to about 250 nucleotides in length. The optimal orpreferred length may vary under different conditions, which can easilybe tested by one of ordinary skill in the art according to the methodsdescribed herein.

The terms “amplicon” and “amplification product” refer to a nucleic acidmolecule generated during an amplification procedure that issubstantially complementary or identical to a sequence contained withinthe target nucleic acid.

As used herein, the term “oligonucleotide” or “oligo” or “oligomer” isintended to encompass a singular “oligonucleotide” as well as plural“oligonucleotides,” and refers to any polymer of two or more ofnucleotides, nucleosides, nucleobases or related compounds used as areagent in the amplification methods of the present invention, as wellas subsequent detection methods. The oligonucleotide may be DNA and/orRNA and/or analogs thereof. The term oligonucleotide does not denote anyparticular function to the reagent; rather, it is used generically tocover all such reagents described herein. An oligonucleotide may servevarious different functions, e.g., target capture oligomers hybridize totarget nucleic acids for capture and isolation of nucleic acids;amplification oligomer include heterologous amplification oligomers,primer oligomers and promoter-based amplification oligomers; primeroligomers hybridize to complementary strands and are then extended inthe presence of a nucleic acid polymerase; alternatively, primersfurther a promoter sequence recognized by an RNA polymerase that allowsfor transcription, and oligomers may function to prevent hybridizationor impede primer extension if appropriately situated and/or modified.Specific oligonucleotides of the present invention are described herein.As used herein, an oligonucleotide can be virtually any length, limitedonly by its specific function in the amplification reaction or indetecting an amplification product of the amplification reaction.

Oligonucleotides of a defined sequence and chemical structure may beproduced by techniques known to those of ordinary skill in the art, suchas by chemical or biochemical synthesis, and by in vitro or in vivoexpression from recombinant nucleic acid molecules, e.g., bacterial orviral vectors. As intended by this disclosure, an oligonucleotide doesnot consist solely of wild-type chromosomal DNA or the in vivotranscription products thereof.

Oligonucleotides may be modified in any way, as long as a givenmodification is compatible with the desired function of a givenoligonucleotide. One of ordinary skill in the art can easily determinewhether a given modification is suitable or desired for any givenoligonucleotide of the present invention. Modifications include basemodifications, sugar modifications or backbone modifications. Basemodifications include, but are not limited to the use of the followingbases in addition to adenine, cytidine, guanosine, thymine and uracil:C-5 propyne, 2-amino adenine, 5-methyl cytidine, inosine, and dP and dKbases. The sugar groups of the nucleoside subunits may be ribose,deoxyribose and analogs thereof, including, for example, ribonucleosideshaving a 2′-O-methyl (2′-O-Me) substitution to the ribofuranosyl moiety.See Becker et al., U.S. Pat. No. 6,130,038. Other sugar modificationsinclude, but are not limited to 2′-amino, 2′-fluoro,(L)-alpha-threofuranosyl, and pentopuranosyl modifications. Thenucleoside subunits may by joined by linkages such as phosphodiesterlinkages, modified linkages or by non-nucleotide moieties that do notprevent hybridization of the oligonucleotide to its complementary targetnucleic acid sequence. Modified linkages include those linkages in whicha standard phosphodiester linkage is replaced with a different linkage,such as a phosphorothioate linkage or a methylphosphonate linkage. Thenucleobase subunits may be joined, for example, by replacing the naturaldeoxyribose phosphate backbone of DNA with a pseudo peptide backbone,such as a 2-aminoethylglycine backbone that couples the nucleobasesubunits by means of a carboxymethyl linker to the central secondaryamine. (DNA analogs having a pseudo peptide backbone are commonlyreferred to as “peptide nucleic acids” or “PNA” and are disclosed byNielsen et al., “Peptide Nucleic Acids,” U.S. Pat. No. 5,539,082.) Otherlinkage modifications include, but are not limited to, morpholino bonds.

Non-limiting examples of oligonucleotides or oligomers contemplated bythe present invention include nucleic acid analogs containing bicyclicand tricyclic nucleoside and nucleotide analogs (LNAs). See Imanishi etal., U.S. Pat. No. 6,268,490; and Wengel et al., U.S. Pat. No.6,670,461.) Any nucleic acid analog is contemplated by the presentinvention provided the modified oligonucleotide can perform its intendedfunction, e.g., hybridize to a target nucleic acid under stringenthybridization conditions, self-hybridize under appropriate conditions toform a hairpin structure, synthesize complementary nucleic acids strandsif a target nucleic acid sequence under amplification conditions, orinteract with a DNA or RNA polymerase, thereby initiating extension ortranscription.

While design and sequence of oligonucleotides for the present inventiondepend on their function as described below, several variables mustgenerally be taken into account. Among the most critical are: length,melting temperature (Tm), specificity, complementarity with otheroligonucleotides in the system, G/C content, polypyrimidine (T, C) orpolypurine (A, G) stretches, and the 3′-end sequence. Controlling forthese and other variables is a standard and well-known aspect ofoligonucleotide design, and various computer programs are readilyavailable to screen large numbers of potential oligonucleotides foroptimal ones.

The 3′-terminus of an oligonucleotide (or other nucleic acid) can beblocked in a variety of ways using a blocking moiety, as describedbelow. A “blocked” oligonucleotide is not efficiently extended by theaddition of nucleotides to its 3′-terminus, by a DNA- or RNA-dependentDNA polymerase, to produce a complementary strand of DNA. As such, a“blocked” oligonucleotide cannot be considered a “primer.”

As used in this disclosure, the phrase “an oligonucleotide having anucleic acid sequence ‘comprising,’ ‘consisting of,’ or ‘consistingessentially of’ a sequence selected from” a group of specific sequencesmeans that the oligonucleotide, as a basic and novel characteristic, iscapable of stably hybridizing to a nucleic acid having the exactcomplement of one of the listed nucleic acid sequences of the groupunder stringent hybridization conditions. An exact complement includesthe corresponding DNA or RNA sequence.

The phrase “an oligonucleotide substantially corresponding to a nucleicacid sequence” means that the referred to oligonucleotide issufficiently similar to the reference nucleic acid sequence such thatthe oligonucleotide has similar hybridization properties to thereference nucleic acid sequence in that it would hybridize with the sametarget nucleic acid sequence under stringent hybridization conditions.

One skilled in the art will understand that “substantiallycorresponding” oligonucleotides can vary from the referred to sequenceand still hybridize to the same target nucleic acid sequence. Variationscan be in the form of one or more nucleotide deletions, additions,substitutions or modifications in the oligomer sequence compared to thatoligomers intended target sequence. This variation from the nucleic acidmay be stated in terms of a percentage of identical bases within thesequence or the percentage of perfectly complementary bases betweenoligomer and its target sequence. Thus, an oligonucleotide of thepresent invention substantially corresponds to a reference nucleic acidsequence if these percentages of base identity or complementarity arefrom 100% to about 80%. In preferred embodiments, the percentage is from100% to about 85%. In more preferred embodiments, this percentage can befrom 100% to about 90%; in other preferred embodiments, this percentageis from 100% to about 95%. One skilled in the art will understand thevarious modifications to the hybridization conditions that might berequired at various percentages of complementarity to allowhybridization to a specific target sequence without causing anunacceptable level of non-specific hybridization.

The “tag-closing sequence” present in the inactivatable target captureoligomer may be essentially any heterologous sequence provided that itdoes not stably hybridize to the target nucleic acid sequence ofinterest and, thereby, participate in selective hybridization andcapture. The tag-closing sequence preferably does not stably hybridizeto any sequence derived from the genome of an organism being tested or,more particularly, to any target nucleic acid under reaction conditions.A tag-closing sequence that is present in a inactivatable target captureoligonucleotide is preferably designed so as not to substantially impairor interfere with the ability of the target hybridizing sequence tohybridize to its target sequence. Moreover, in certain alternativeembodiments wherein the tag-closing sequence is also a tag sequence itis of sufficient length and composition such that once the tag sequenceor a complement thereof has been incorporated into an initial DNA primerextension product, a tag-specific priming oligonucleotide can then beused to participate in subsequent rounds of amplification as describedherein. A tag-closing sequence of the present invention is typically atleast 3 nucleotides in length, and may extend up to 6, 10, 15, 20, 25,30, 35, 40, 50 or more nucleotides in length. Skilled artisans willrecognize that the design of tag-closing sequences and, optionally, tagsequences for use in the present invention can follow any of a number ofsuitable strategies, while still achieving the objectives and advantagesdescribed herein.

The term “inactivating” means that a target hybridizing region isaltered so that it will not stably bind to a target nucleic acidsequence under target hybridization conditions. Thus, under a first setof conditions, an inactivatable target capture oligomer, and,optionally, a heterologous amplification oligomer will hybridize totarget nucleic acids present in the sample. Unhybridized inactivatabletarget capture oligomer are inactivated under a second set of conditionsin which the inactivatable target capture oligomer is configured toself-hybridize into an inactive and preferably hairpin configuration.The target hybridizing regions are then sterically blocked, whichprevents or substantially reduces binding of inactivated inactivatabletarget capture oligomers to non-target nucleic acids and/orcontaminating nucleic acids. So, “inactivating” means that theinactivatable target capture oligomer is altered from an “active”confirmation which permits the target hybridizing sequence to hybridizeto the target nucleic acid sequence to an “inactive” confirmation whichblocks or otherwise prevents the target hybridizing sequence fromhybridizing to the target nucleic acid sequence. As example only, aninactive confirmation may be formed under stringency conditionspermitting the tag-closing sequence to form a stable hybrid with thetarget hybridizing sequence (e.g., under less stringent conditions thanthe conditions for forming an active confirmation of the inactivatabletarget capture oligonucleotide). Alternatively, a duplex formed betweenthe tag-closing sequence and the target hybridizing sequence may bealtered by an enzyme, such as a DNAse, an S1 nuclease, an endonuclease,such as a restriction enzyme which cleaves a double-stranded restrictionsite formed between the tag-closing sequence and the target hybridizingsequence, a ribonuclease activity (e.g., RNAse H activity) for digestingthe RNA component (e.g., target hybridizing sequence) of a DNA:RNAhybrid, or an exonuclease having a 3′-to-5′ or 5′-to-3′ activity forremoving nucleotides from the target hybridizing sequence hybridized tothe tag-closing sequence. However, to avoid exposing a sample to apotentially contaminating source of the target nucleic acid sequence,the use of enzymes to inactivate inactivatable target captureoligonucleotides that have not hybridized to the target nucleic acidsequence is generally not preferred. Other inactivating means includechemicals for altering the target hybridizing sequence so that it isincapable of hybridizing to a target nucleic acid sequence underamplification conditions. Preferably, but not necessarily, a targethybridizing region of an inactivatable target capture oligomer is 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobasesin length. Preferably, but not necessarily, a tag-closing region of aninactivatable target capture oligomer is 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleobases in length. Someexemplary and non-limiting embodiments of inactivatable target captureoligomers in the current disclosure comprise target hybridizing regionsthat are 17 nucleobases in length, and comprise tag-closing regions thatare 6, 7, 8, 9, 10, 12 and 14 nucleobases in length. In theseembodiments, the binding pair members are substantially homopolymericnucleotide sequences of 33 nucleobases in length. These are non-limitingembodiments provided for example of the current invention.

Moieties can be included in the tag hybridizing sequence to furtherstabilize hybrids formed between the tag-closing sequence and the targethybridizing sequence of inactivatable target capture oligomers,especially where it is anticipated that at least some of the inactiveinactivatable target capture oligomer will be introduced into theamplification reaction mixture. Suitable moieties include modifiednucleotides, including LNAs, 2′-O-Me ribonucleotides, 2,6 diaminopurine. 5-methyl cytosine, and C-5 propynyl cytosine or uracil. Thoseskilled in the art will be able to readily select the number andpositions of such modified nucleotides to limit breathing at the 5′- and3′-ends of the tag-closing sequence and to achieve a desired meltingtemperature of the hybrid without engaging in undue experimentation.Other suitable moieties include minor groove binders and pendant groups,such as purine, DABCYL, pyrine and 5′-trimethoxy stilbene CAP.

As used herein, the term “removing” refers to the physical separation ofcaptured target nucleic acid sequences from unhybridized inactivatabletarget capture oligomer. Captured target nucleic acid sequences can bephysically separated from unhybridized inactivatable target captureoligomer present in a nucleic acid sample by a variety of techniquesknown to those skilled in the art. By way of example, captured targetnucleic acid sequences can be bound to a solid support and immobilizedin a nucleic acid sample while unbound material is removed. To removeunbound material, the solid support can be subjected to one or morewash/rinse steps. A rinse step is typically included where the washsolution contains a component that is inhibitory to subsequent analysisof the target nucleic acids when present at a sufficiently highconcentration, such as a detergent. The solid support preferably bindsspecifically to captured target nucleic acids, to prevent unhybridizedinactivatable target capture oligomer from entering into theamplification reaction. Exemplary means for capturing, immobilizing andpurifying target nucleic acids are discussed below, an example of whichis disclosed by Weisburg et al., U.S. Pat. No. 6,534,273.

The phrases “tag-closing sequence,” “tag closing region” and“tag-closing oligonucleotide” refer to a nucleotide sequence that iscomplementary to a portion of the target hybridizing sequence of aninactivatable target capture oligomer. The length and sequence of thetag-closing sequence are selected so that the tag-closing sequence doesnot stably hybridize to the target hybridizing sequence of theinactivatable target capture oligomer under a first set of conditionspermitting stable hybridization of the target hybridizing sequence to atarget sequence. The tag-closing sequence may include abasic nucleotidesor base mismatches with the target hybridizing sequence. Provided theinactivatable target capture oligomer is not hybridized to the targetsequence, the tag-closing sequence stably hybridizes to the targethybridizing sequence under a second set of less stringent conditions,thus “inactivating” or blocking the inactivatable target captureoligomer from hybridizing to the target sequence. The tag-closingsequence may be in the form of a discrete oligonucleotide, asexemplified in FIGS. 11, 12 & 22, or it may be an integral part of theinactivatable oligomer, so that it forms a hairpin structure under thesecond set of conditions, as exemplified in FIGS. 13, 14 & 23-27. If thetag-closing sequence is joined to the inactivatable oligonucleotide viaa non-nucleotide linker region (e.g., abasic nucleotides or polyethyleneglycol), then the non-nucleotide linker is of sufficient length for thetag-closing sequence to hybridize to the target hybridizing sequenceunder the second set of conditions. The association kinetics are bestwhen the tag-closing sequence and the target hybridizing sequence of thetagged oligonucleotide are contained in the same molecule (integralconfiguration). Under selective conditions, the tag-closing sequence canhybridize to a target hybridizing sequence in an antiparallelorientation, as shown in FIGS. 2, 4, 6, 8, 10, 11, 12, 15 and 16, or ina parallel orientation, as shown in FIGS. 13 and 14. If the tag-closingsequence is a discrete molecule, as illustrated in FIGS. 11, 12 and 22,or joined to the tagged oligonucleotide by a non-nucleotide linker, asillustrated in FIGS. 2, 4, 6, 8, 10, 15, 16 and 23, then the tag-closingsequence is preferably modified to prevent primer extension by a DNApolymerase, such as by positioning a blocking moiety at its 3′-terminus.Suitable blocking moieties are described herein. When hybridized in anantiparallel orientation, as illustrated in FIGS. 13 and 14, the3-terminal base of the tag-closing sequence is preferably hybridized tothe 3-terminal base of the target hybridizing sequence. More preferably,the tag-closing sequence is modified to prevent primer extension by aDNA polymerase. The tag-closing sequence may be modified to prevent theinitiation of DNA synthesis therefrom, which can include a blockingmoiety situated at its 3-terminus. The tag-closing sequence is at least3 nucleobases in length. Typical tag-closing sequences are preferablyfrom 6 to 16 bases in length.

FIG. 22 shows a tag-closing sequence that is a discrete molecule, whileFIGS. 23, 24 and 25 show a capture probe molecule that incorporates thetag-closing sequence as an integral part of the target capture oligomer.This figure illustrates for all embodiments that the discretetag-closing region is capable of stable hybridization to the targethybridizing sequence under conditions of reduced stringency. FIGS. 24and 25 illustrate a tag-closing sequence that is directly joined to thetarget hybridizing sequence. FIG. 23 illustrates a tag-closing sequencethat is indirectly joined to the target hybridizing sequence (e.g.,nucleotide sequence or non-nucleotide linker). In general, a preferredtag-closing sequence hybridizes to 5 to 20 contiguous or non-contiguousbases of the target hybridizing sequence and is from 5 to 20 bases inlength. Preferably, any terminal 3′-ends that can be used for aprimer-based amplification are blocked to prevent such an extensionreaction.

By “amplification” or “nucleic acid amplification” is meant productionof multiple copies of a target nucleic acid that contains at least aportion of the intended specific target nucleic acid sequence. Themultiple copies may be referred to as amplicons or amplificationproducts. In certain embodiments, the amplified target contains lessthan the complete target gene sequence (introns and exons) or anexpressed target gene sequence (spliced transcript of exons and flankinguntranslated sequences). For example, specific amplicons may be producedby amplifying a portion of the target polynucleotide by usingamplification primers that hybridize to, and initiate polymerizationfrom, internal positions of the target polynucleotide. Preferably, theamplified portion contains a detectable target sequence that may bedetected using any of a variety of well-known methods.

Many well-known methods of nucleic acid amplification requirethermalcycling to alternately denature double-stranded nucleic acids andhybridize primers; however, other well-known methods of nucleic acidamplification are isothermal. PCR and RT-PCR are thermalcyclingtechniques that use multiple cycles of denaturation, annealing of primerpairs to opposite strands, and primer extension to exponentiallyincrease copy numbers of the target sequence. Variations of the PCRtechnique include SDA and tSDA. Isothermal techniques include, but arenot limited to, NASBA, Q.beta. replicase and transcription-basedamplification methods like self-sustained sequence replication and TMA.Other illustrative amplification methods suitable for use in accordancewith the present invention include, but are not limited to, RCA, had,and LAMP.

TMA employs an RNA polymerase to produce multiple RNA transcripts of atarget region (e.g., Kacian et al., U.S. Pat. Nos. 5,480,784 and5,399,491; and Becker et al., U.S. Pub. No. US 2006-0046265 A1). TMAuses at least one “promoter oligonucleotide” or “promoter-primer” thathybridizes to a target nucleic acid in the presence of a reversetranscriptase and an RNA polymerase to form a double-stranded promoterfrom which the RNA polymerase produces RNA transcripts. Thesetranscripts can become templates for further rounds of TMA in thepresence of a second primer capable of hybridizing to the RNAtranscripts. Unlike PCR, LCR or other methods that require heatdenaturation, TMA is an isothermal method that uses an RNAse H activityto digest the RNA strand of an RNA:DNA hybrid, thereby making the DNAstrand available for hybridization with a primer or promoter-primer.Generally, the RNAse H activity associated with the reversetranscriptase provided for amplification is used.

In one illustrative TMA method, one amplification primer is anoligonucleotide promoter-primer that comprises a promoter sequence whichbecomes functional when double-stranded, located 5′ of a target-bindingsequence, which is capable of hybridizing to a binding site of a targetRNA at a location 3′ to the sequence to be amplified. A promoter-primermay be referred to as a “T7-primer” when it is specific for T7 RNApolymerase recognition. Under certain circumstances, the 3′ end of apromoter-primer, or a subpopulation of such promoter-primers, may bemodified to block or reduce primer extension. From an unmodifiedpromoter-primer, reverse transcriptase creates a cDNA copy of the targetRNA, while RNAse H activity degrades the target RNA. A secondamplification primer then binds to the cDNA. This primer may be referredto as a “non-T7 primer” to distinguish it from a “T7-primer”. From thissecond amplification primer, reverse transcriptase creates another DNAstrand, resulting in a double-stranded DNA with a functional promoter atone end. When double-stranded, the promoter sequence is capable ofbinding an RNA polymerase to begin transcription of the target sequenceto which the promoter-primer is hybridized. An RNA polymerase uses thispromoter sequence to produce multiple RNA transcripts (i.e., amplicons),generally about 100 to 1,000 copies. Each newly synthesized amplicon cananneal with the second amplification primer. Reverse transcriptase canthen create a DNA copy, while the RNAse H activity degrades the RNA ofthis RNA:DNA duplex. The promoter-primer can then bind to the newlysynthesized DNA, allowing the reverse transcriptase to create adouble-stranded DNA, from which the RNA polymerase produces multipleamplicons. Thus, a billion-fold isothermic amplification can be achievedusing two amplification primers.

In another illustrative TMA method, one or more features as described inBecker et al., U.S. Pub. No. US 2006-0046265 are optionallyincorporated. Preferred TMA methods in this respect include the use ofblocking moieties, terminating moieties, and other modifying moietiesthat provide improved TMA process sensitivity and accuracy. Thus,certain preferred embodiments of the present invention employinactivatable oligonucleotides, as described herein, in conjunction withthe methods as described in Becker et al., U.S. Pub. No. US2006-0046265.

By “detectable amplification” is meant that a detectable signalassociated with an amplification product in an amplification reactionmixture rises above a predetermined background or threshold level(end-point amplification) or rises above a background or threshold levelwithin a predetermined period of time (real-time amplification). See,e.g., Light et al., U.S. Pub. No. US 2006-0276972, paragraphs 506-549.The amplification product contains a sequence having sequence identitywith a target nucleic acid sequence or its complement and can bedetected with, for example, an intercalating dye or a detection probehaving specificity for a region of the target nucleic acid sequence orits complement.

An amplification product can be detected by any conventional means. Forexample, amplification product can be detected by hybridization with adetectably labeled probe and measurement of the resulting hybrids.Design criteria in selecting probes for detecting particular targetsequences are well known in the art and are described in, for example,Hogan et al., U.S. Pat. No. 6,150,517. Amplification products can beassayed by the Hybridization Protection Assay (“HPA”), which involveshybridizing a chemiluminescent oligonucleotide probe to the targetsequence, e.g., an acridinium ester-labeled (“AE”) probe, selectivelyhydrolyzing the chemiluminescent label present on unhybridized probe,and measuring the chemiluminescence produced from the remaining probe ina luminometer. See, e.g., Arnold et al., U.S. Pat. No. 5,283,174 andNelson et al., Nonisotopic Probing, Blotting and Sequencing, ch. 17(Larry J. Kricka ed., 2d ed. 1995).

Further embodiments provide quantitative evaluation of the amplificationprocess in real-time by methods described herein. Evaluation of anamplification process in “real-time” involves determining the amount ofamplicon in the reaction mixture either continuously or periodicallyduring the amplification reaction, and the determined values are used tocalculate the amount of target sequence initially present in the sample.There are a variety of methods for determining the amount of initialtarget sequence present in a sample based on real-time amplification.These include those disclosed by Wittwer et al., U.S. Pat. No.6,303,305, and Yokoyama et al., U.S. Pat. No. 6,541,205. Another methodfor determining the quantity of target sequence initially present in asample, but which is not based on a real-time amplification, isdisclosed by Ryder et al., U.S. Pat. No. 5,710,029.

Amplification products may be detected in real-time through the use ofvarious self-hybridizing probes, most of which have a stem-loopstructure. Such self-hybridizing probes are labeled so that they emitdifferently detectable signals, depending on whether the probes are in aself-hybridized state or an altered state through hybridization to atarget sequence. By way of example, “molecular torches” are a type ofself-hybridizing probe that includes distinct regions ofself-complementarity (referred to as “the target binding domain” and“the target closing domain”), which are connected by a joining region(e.g., non-nucleotide linker) and which hybridize to each other underpredetermined hybridization assay conditions. In a preferred embodiment,molecular torches contain single-stranded base regions in the targetbinding domain that are from 1 to about 20 bases in length and areaccessible for hybridization to a target sequence present in anamplification product under strand displacement conditions. Under stranddisplacement conditions, hybridization of the two complementary regions(which may be fully or partially complementary) of the molecular torchis favored, except in the presence of the target sequence, which willbind to the single-stranded region present in the target binding domainand displace all or a portion of the target closing domain. The targetbinding domain and the target closing domain of a molecular torchinclude a detectable label or a pair of interacting labels (e.g.,luminescent/quencher) positioned so that a different signal is producedwhen the molecular torch is self-hybridized than when the moleculartorch is hybridized to the target sequence, thereby permitting detectionof probe:target duplexes in a test sample in the presence ofunhybridized molecular torches. Molecular torches and a variety of typesof interacting label pairs are disclosed by Becker et al., U.S. Pat. No.6,534,274.

Another example of a detection probe having self-complementarity is a“molecular beacon.” Molecular beacons include nucleic acid moleculeshaving a target complement sequence, an affinity pair (or nucleic acidarms) holding the probe in a closed conformation in the absence of atarget sequence present in an amplification product, and a label pairthat interacts when the probe is in a closed conformation. Hybridizationof the target sequence and the target complement sequence separates themembers of the affinity pair, thereby shifting the probe to an openconformation. The shift to the open conformation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beaconsare disclosed by Tyagi et al., U.S. Pat. Nos. 5,925,517, and 6,150,097.

Other self-hybridizing probes for use in the present invention are wellknown to those of ordinary skill in the art. By way of example, probebinding pairs having interacting labels, such as those disclosed byMorrison, U.S. Pat. No. 5,928,862 and Gelfand et al., U.S. Pat. No.5,804,375, might be adapted for use in the present invention. Additionaldetection systems include “molecular switches,” Arnold et al., U.S. Pub.No. U.S. 2005-0042638 A1. And other probes, such as those comprisingintercalating dyes and/or fluorochromes, might be useful for detectionof amplification products in the present invention. See, e.g., Ishiguroet al., U.S. Pat. No. 5,814,447.

In amplification and detection methods where the initial target sequenceand the RNA transcription product share the same sense, it may bedesirable to initiate amplification before adding probe for real-timedetection. Adding probe prior to initiating an amplification reactionmay slow the rate of amplification since probe which binds to theinitial target sequence has to be displaced or otherwise remove duringthe primer extension step to complete a primer extension product havingthe complement of the target sequence. The initiation of amplificationis judged by the addition of amplification enzymes (e.g., a reversetranscriptase and an RNA polymerase).

“Selective amplification” as used herein, refers to the amplification ofa target nucleic acid sequence that has been selectively hybridized andcaptured using compositions and methods according to the presentinvention, and where detectable amplification is limited orsubstantially limited to amplification of target sequence and is notcontributed by non-target nucleic acid sequence and/or contaminantnucleic acid. As used herein, a “non-target nucleic acid” is a nucleicacid that is present in a reaction mixture but is not the desirednucleic acid. Non-target nucleic acids include, but are not limited to,nucleic acids present in a whole-cell lysate other than the nucleic acidof interest. For example, if the target nucleic acids is a micro RNAknown or thought to be present in a cell sample, the remaining nucleicacid present in that sample (or lysate thereof) are the non-targetnucleic acids. Here, the inactivatable target capture oligomer isconfigured to comprise a target hybridizing region substantiallycomplementary to this small target nucleic acid. It is desirous that theinactivatable target capture oligomer does not hybridize any non-targetnucleic acid, which are hindrances to downstream analysis of the desiredtarget nucleic acids. Thus, an active form of the inventioninactivatable target capture oligomer is allowed to hybridize the targetnucleic acid under a first set of conditions biasing the captureoligomer to the target, and then the inactivatable target captureoligomer is made inactive to prevent binding to non-target nucleicacids. As is used herein, a “contaminant nucleic acid” is a nucleic acidthat is introduced into a system from an outside source. Such sourcesinclude, but are not limited to, reagent preparations, labwares, labpersonnel and laboratory workspace.

By “amplification conditions” is meant conditions permitting nucleicacid amplification. Amplification conditions may, in some embodiments,be less stringent than “stringent hybridization conditions” as describedherein. Oligonucleotides used in amplification reactions hybridize totheir intended targets under amplification conditions, but may or maynot hybridize under stringent hybridization conditions. On the otherhand, detection probes are typically designed to hybridize understringent hybridization conditions. While the Examples section infraprovides preferred amplification conditions for amplifying targetnucleic acid sequences, other acceptable conditions to carry out nucleicacid amplifications could be easily ascertained by someone havingordinary skill in the art depending on the particular method ofamplification employed.

Nucleic acid hybridization is the process by which two nucleic acidstrands having completely or partially complementary nucleotidesequences come together under predetermined reaction conditions to forma stable, double-stranded hybrid. Either nucleic acid strand may be adeoxyribonucleic acid (DNA) or a ribonucleic acid (RNA) or analogsthereof. Thus, hybridization can involve RNA:RNA hybrids, DNA:DNAhybrids, RNA:DNA hybrids, or analogs thereof. The two constituentstrands of this double-stranded structure, sometimes called a hybrid,are held together by hydrogen bonds. Although these hydrogen bonds mostcommonly form between nucleotides containing the bases adenine andthymine or uracil (A and T or U) or cytosine and guanine (C and G) onsingle nucleic acid strands, base pairing can also form between basesthat are not members of these “canonical” pairs. Non-canonical basepairing is well known in the art. (See, e.g., Adams, et al., TheBiochemistry of the Nucleic Acids, (11th ed. 1992).)

“Hybridization conditions” refer to the cumulative physical and chemicalconditions under which nucleic acid sequences that are completely orpartially complementary form a hybridization duplex or complex. Suchconditions are well known to those skilled in the art, are predictablebased on sequence composition of the nucleic acids involved inhybridization, or may be determined empirically by using routine testing(e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed.(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) at§§ 1.90-1.91, 7.37-7.57, 9.47-9.51, 11.12-11.13, and 11.45-11.57).

“Stringent hybridization conditions” or “stringent conditions” refer toconditions where a specific nucleic acid strand (including, but notlimited to, an inactivatable target capture oligomer) is able topreferentially hybridize with a target nucleic acid over other nucleicacids present in the test sample. It will be appreciated that theseconditions may vary depending upon factors including the GC content andlength of the nucleic acid sequences, the hybridization temperature, thecomposition of the hybridization reagent or solution, and the degree ofhybridization specificity sought. Some specific stringent hybridizationconditions are provided in the disclosure below.

By “nucleic acid hybrid” or “hybrid” or “duplex” is meant a nucleic acidstructure containing a double-stranded, hydrogen-bonded region whereeach strand is complementary to the other, and where the region issufficiently stable under stringent hybridization conditions to bedetected by means including, but not limited to, chemiluminescent orfluorescent light detection, autoradiography, or gel electrophoresis.Such hybrids may comprise RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

By “complementary” is meant that the nucleotide sequences of similarregions of two single-stranded nucleic acids, or to different regions ofthe same single-stranded nucleic acid have a nucleotide base compositionthat allow the single-stranded regions to hybridize together in astable, double-stranded hydrogen-bonded region under stringenthybridization or amplification conditions. When a contiguous sequence ofnucleotides of one single-stranded region is able to form a series of“canonical” hydrogen-bonded base pairs with an analogous sequence ofnucleotides of the other single-stranded region, such that A is pairedwith U or T and C is paired with G, the nucleotides sequences are“perfectly” complementary.

By “preferentially hybridize” is meant that under stringenthybridization conditions, certain complementary nucleotides ornucleobase sequences hybridize to form a stable hybrid preferentiallyover other, less stable duplexes. By “does not stably hybridize” ismeant that a stable hybrid is not formed in appreciable and/ordetectable amounts under a defined set of conditions. As a non-limitingexample, a probe hybridizes to a target sequence or replicate thereof toa sufficiently greater extent than to a non-target sequence, thus enableone having ordinary skill in the art to accurately quantitate the RNAreplicates or complementary DNA (cDNA) of the target sequence formedduring the amplification. In a further non-limiting example, a targethybridization region of an inactivatable target capture oligomerhybridizes to a target nucleic acid to a sufficiently greater extentthan to a non-target or contaminating nucleic acid sequence.

By “stable” or “stably hybridize” is meant that the temperature of areaction mixture is at least 2.deg.C below the melting temperature of anucleic acid duplex.

The term “amplification oligomer” is used herein to refer to oligomersused as primers, promoter-based amplification oligomers such as promoterprimers and promoter providers, and heterologous amplificationoligomers.

A “heterologous amplification oligomer” comprises a first region that isa target hybridizing region and a second region that is a tag sequenceregion. The tag sequence of a heterologous amplification oligomer isconfigured so that under conditions wherein the target hybridizingregion stably hybridizes with a target nucleic acid sequence, this tagsequence does not stably hybridize with the target nucleic acid or anyknown non-target or contaminating nucleic acid sequences believed to bein a sample. Following a first nucleic acid synthesis event using theheterologous amplification oligomer as a primer, the tag sequence formsan integral part of the resultant amplicon sequence and any subsequentcomplimentary or copy strands made thereof. Second round amplificationis then performed using an amplification oligomer that targets this tagsequence, or complement thereof, thereby reducing or eliminatingamplification of non-target and contaminating nucleic acids.Heterologous amplification oligomers can be promoter-based oligomers aswell, wherein a third region is included; said region being an RNApolymerase promoter sequence. Such a heterologous amplification oligomeroperates substantially as described directly above if the heterologousamplification oligomer is a promoter primer. In this configuration,though, an RNA copy containing the tag sequence or its complement istranscribed from a template. If, though, the heterologous amplificationoligomer is a promoter provider, then an RNA molecule containing thecomplement of the tag sequence is first made, and subsequentamplification using the sequence or its complement takes place fromthere.

As is well known in the art, a “promoter” is a specific nucleic acidsequence that is recognized by a DNA-dependent RNA polymerase(“transcriptase”) as a signal to bind to the nucleic acid and begin thetranscription of RNA at a specific site. Efficient transcription of RNAcan take place under conditions where a double-stranded promoter is notformed through an extension reaction with the template nucleic acid. Thetemplate nucleic acid (the sequence to be transcribed) need not bedouble-stranded. Individual DNA-dependent RNA polymerases recognize avariety of different promoter sequences, which can vary markedly intheir efficiency in promoting transcription. When an RNA polymerasebinds to a promoter sequence to initiate transcription, that promotersequence is not part of the sequence transcribed. Thus, the RNAtranscripts produced thereby will not include that promoter sequence.

According to the present invention, a “promoter oligonucleotide” refersto an oligonucleotide comprising first and second regions, and which isoptionally modified to prevent the initiation of DNA synthesis from its3′-terminus. The “first region” of a promoter oligonucleotide of thepresent invention comprises a target hybridizing region. The targethybridizing region of a promoter oligonucleotide is typically at least10 nucleotides in length, and may extend up to 15, 20, 25, 30, 35, 40,50 or more nucleotides in length. The “second region” comprises apromoter sequence for an RNA polymerase. A promoter oligonucleotide issometimes engineered so that it is incapable of being extended by anRNA- or DNA-dependent DNA polymerase, e.g., reverse transcriptase,preferably comprising a blocking moiety at its 3′-terminus as describedabove. Suitable and preferred promoter oligonucleotides are describedherein. Promoter-based amplification oligomers having a modified3′-terminus are referred to herein as “promoter providers,” while thosewithout such a modification are referred to herein as “promoterprimers.” In one example, at least about 80% of the oligonucleotidespresent in the amplification reaction and comprising a promoter furthercomprise a 3-blocking moiety. In another embodiment, at least about 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% of the oligonucleotides provided tothe amplification reaction comprising a promoter are further modified tocomprise a 3′-blocking moiety. In another embodiment, anyoligonucleotide used in an amplification reaction that comprises apromoter sequence further comprise a 3′-terminus blocking moiety.Assaying promoter oligonucleotides with variations in the promotersequences is easily carried out by the skilled artisan using routinemethods. Furthermore, if it is desired to utilize a different RNApolymerase, then the promoter sequence in the promoter oligonucleotideis easily substituted with a different and more compatible promotersequence. Substituting different promoter sequences is well within theunderstanding and capabilities of those of ordinary skill in the art.

The formation of a double-stranded promoter sequence through extensionof a template nucleic acid is not necessary to permit initiation oftranscription of RNA complementary to the first DNA primer extensionproduct. The resulting “first” RNA products are substantially identicalto the target sequence, having a 5′-end defined by the transcriptioninitiation point, and a 3′-end defined by the 5′-end of the first DNAprimer extension product. A sufficient number of first RNA products areproduced to automatically recycle in the system without furthermanipulation. The priming oligonucleotide hybridizes to the 3′-end ofthe first RNA products, and is extended by a DNA polymerase to form asecond DNA primer extension product. Unlike the first DNA primerextension product formed without the use of a terminatingoligonucleotide or other binding molecule, the second DNA primerextension product has a defined 3′-end which is complementary to the5′-ends of the first RNA products. The second DNA primer extensionproduct is separated (at least partially) from the RNA template using anenzyme that selectively degrades the RNA template. The single-strandedsecond DNA primer extension product is then treated with a promoteroligonucleotide as described above, and the second region of thepromoter oligonucleotide acts as a template, allowing the second DNAprimer extension product to be further extended to add a base regioncomplementary to the second region of the promoter oligonucleotide,i.e., the region comprising the promoter sequence, rendering thepromoter double-stranded. An RNA polymerase that recognizes the promoterbinds to the promoter sequence, and initiates transcription of multiple“second” RNA products complementary to the second DNA primer extensionproduct, and substantially identical to the target sequence. The secondRNA transcripts so produced automatically recycle in the above systemwithout further manipulation. Thus, this reaction is autocatalytic.

“Universal” oligonucleotides or “pan” oligonucleotides includeoligonucleotides that can be used in an amplification reaction toidentify the presence of nucleic acid sequences of a class of organismsbased upon highly conserved sequences that are unique to a class oforganisms. (As used herein, the term “class” does not necessarily implya recognized phylogenetic grouping or organisms.) For example, thehighly conserved 16S ribosomal RNA-coding sequences contain regions thatare found in bacteria, or groupings of bacteria (e.g., Eubacteria,Gram-positive bacteria or Gram-negative bacteria), but are not in humansand other higher organisms, and thus oligonucleotides may be designedand used in a nucleic acid amplification reaction to detect the presenceof bacterial sequences in a sample of interest. See, e.g., McCabe et al.(1999) Molecular Genetics and Metabolism 66, 205-211; Schmidt, T. et al.(1994) Meth. Enzymol. 235, 205-222 (method for identifying pathogens);Kunishima, S. et al., (2000) Transfusion 40, 1420 (method for detectingbacteria in blood); Greisen, K. (1994) J. Clin. Microbiol. 32, 335-351(method for detecting pathogenic bacteria in cerebral spinal fluid);Jordan, J. (2005) J. Mol. Diag. 7, 575-581 (method for diagnosing sepsisin neonates); Rothman, R. et al. (2002) J. Infect. Dis. 186, 1677-1681(method for diagnosing acute bacterial endocarditis); and Cox, C. et al.(2002) Arthritis Res. Ther. 5, R1-R8 (detecting bacteria in synovialfluid). Similarly, universal oligonucleotides for other classes oforganisms, such as fungal pathogens, have been described. See, e.g.,Maaroati, Y. et al. (2003) J. Clin. Microbiol. 41, 3293-3298 (method forquantifying Candida albicans in blood); Carr, M. et al. (2005) J. Clin.Microbiol. 43, 3023-3026 (method for detecting Candida dubliniensis inblood); and White, P. et al. (2003) J. Med. Microbiol. 52, 229-238(method for detecting systemic fungal infections). Essentially anyuniversal oligonucleotides known or developed for a given class oforganism may be advantageously employed in the methods described herein.

Apriming oligonucleotide is an oligonucleotide, at least the 3′-end ofwhich is complementary to a nucleic acid template, and which complexes(by hydrogen bonding or hybridization) with the template to give aprimer:template complex suitable for initiation of synthesis by an RNA-or DNA-dependent DNA polymerase. A priming oligonucleotide is extendedby the addition of covalently bonded nucleotide bases to its3′-terminus, which bases are complementary to the template. The resultis a primer extension product. A priming oligonucleotide is typically atleast 10 nucleotides in length, and may extend up to 15, 20, 25, 30, 35,40, 50 or more nucleotides in length. Suitable and preferred primingoligonucleotides are described herein. Virtually all DNA polymerases(including reverse transcriptases) that are known require complexing ofan oligonucleotide to a single-stranded template (“priming”) to initiateDNA synthesis, whereas RNA replication and transcription (copying of RNAfrom DNA) generally do not require a primer. By its very nature of beingextended by a DNA polymerase, a priming oligonucleotide does notcomprise a 3′-blocking moiety.

A “displacer oligonucleotide” is a priming oligonucleotide thathybridizes to a template nucleic acid upstream from a neighboringpriming oligonucleotide hybridized to the 3′-end of a target sequence(referred to herein as the “forward priming oligonucleotide”). By“upstream” is meant that a 3′-end of the displacer oligonucleotidecomplexes with the template nucleic acid 5′ to a 3′-end of the forwardpriming oligonucleotide. When hybridized to the template nucleic acid,the 3′-terminal base of the displacer oligonucleotide is preferablyadjacent to or spaced from the 5-terminal base of the forward primingoligonucleotide. More preferably, the 3′-terminal base of the displaceroligonucleotide is spaced from 5 to 35 bases from the 5′-terminal baseof the forward priming oligonucleotide. The displacer oligonucleotidemay be provided to a reaction mixture contemporaneously with the forwardpriming oligonucleotide or after the forward priming oligonucleotide hashad sufficient time to hybridize to the template nucleic acid. Extensionof the forward priming oligonucleotide can be initiated prior to orafter the displacer oligonucleotide is provided to a reaction mixture.Under amplification conditions, the displacer oligonucleotide isextended in a template-dependent manner, thereby displacing a primerextension product comprising the forward priming oligonucleotide that iscomplexed with the template nucleic acid. Once displaced from thetemplate nucleic acid, the primer extension product comprising theforward priming oligonucleotide is available for complexing with apromoter oligonucleotide. The forward priming oligonucleotide and thedisplacer oligonucleotide both preferentially hybridize to the targetnucleic acid. Examples of displacer oligonucleotides and their uses aredisclosed by Becker et al., U.S. Pub. No. US 20070202523 A1, commonlyowned herewith.

As used herein, a “blocking moiety” is a substance used to “block” the3′-terminus of an oligonucleotide or other nucleic acid so that itcannot be efficiently extended by a nucleic acid polymerase. A blockingmoiety may be a small molecule, e.g., a phosphate or ammonium group, orit may be a modified nucleotide, e.g., a 3′2′ dideoxynucleotide or 3′deoxyadenosine 5′-triphosphate (cordycepin), or other modifiednucleotide. Additional blocking moieties include, for example, the useof a nucleotide or a short nucleotide sequence having a 3′-to-5′orientation, so that there is no free hydroxyl group at the 3′-terminus,the use of a 3′ alkyl group, a 3′ non-nucleotide moiety (see, e.g.,Arnold et al., U.S. Pat. No. 6,031,091), phosphorothioate, alkane-diolresidues, peptide nucleic acid (PNA), nucleotide residues lacking a 3′hydroxyl group at the 3′-terminus, or a nucleic acid binding protein.Preferably, the 3′-blocking moiety comprises a nucleotide or anucleotide sequence having a 3′-to-5′ orientation or a 3′ non-nucleotidemoiety, and not a 3′2′-dideoxynucleotide or a 3′ terminus having a freehydroxyl group. Additional methods to prepare 3′-blockingoligonucleotides are well known to those of ordinary skill in the art.

As used herein, a “binding molecule” is a substance which hybridizes toor otherwise binds to an RNA target nucleic acid adjacent to or near the5′-end of the desired target sequence, so as to limit a DNA primerextension product to a desired length, i.e., a primer extension producthaving a generally defined 3′-end. As used herein, the phrase “defined3′-end” means that the 3′-end of a primer extension product is notwholly indeterminate, as would be the case in a primer extensionreaction which occurs in the absence of a binding molecule, but ratherthat the 3′-end of the primer extension product is generally known towithin a small range of bases. In certain embodiments, a bindingmolecule comprises a base region. The base region may be DNA, RNA, aDNA:RNA chimeric molecule, or an analog thereof. Binding moleculescomprising a base region may be modified in one or more ways, asdescribed herein. Exemplary base regions include terminating anddigestion oligonucleotides, as described below. In other embodiments, abinding molecule may comprise, for example, a protein or drug capable ofbinding RNA with sufficient affinity and specificity to limit a DNAprimer extension product to a pre-determined length.

As is used herein, a “terminating oligonucleotide” is an oligonucleotidecomprising a base sequence that is complementary to a region of a targetnucleic acid sequence in the vicinity of the 5′-end of the targetsequence, so as to “terminate” primer extension of a nascent nucleicacid that includes a priming oligonucleotide, thereby providing adefined 3′-end for the nascent nucleic acid strand. A terminatingoligonucleotide is designed to hybridize to the target nucleic acid at aposition sufficient to achieve the desired 3′-end for the nascentnucleic acid strand. The positioning of the terminating oligonucleotideis flexible depending upon its design. A terminating oligonucleotide maybe modified or unmodified. In certain embodiments, terminatingoligonucleotides are synthesized with at least one or more 2′-O-Meribonucleotides. These modified nucleotides have demonstrated higherthermal stability of complementary duplexes. The 2′-O-Me ribonucleotidesalso function to increase the resistance of oligonucleotides toexonucleases, thereby increasing the half-life of the modifiedoligonucleotides. See, e.g., Majlessi et al. (1988) Nucleic Acids Res.26, 2224-9. Other modifications as described elsewhere herein may beutilized in addition to or in place of 2′-O-Me ribonucleotides. Forexample, a terminating oligonucleotide may comprise PNA or an LNA. See,e.g., Petersen et al. (2000) J. Mol. Recognit. 13, 44-53. A terminatingoligonucleotide typically includes a blocking moiety at its 3′-terminusto prevent extension. A terminating oligonucleotide may also comprise aprotein or peptide joined to the oligonucleotide so as to terminatefurther extension of a nascent nucleic acid chain by a polymerase. Aterminating oligonucleotide is typically at least 10 bases in length,and may extend up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides inlength. Suitable and preferred terminating oligonucleotides aredescribed herein. It should be noted that while a terminatingoligonucleotide typically or necessarily includes a 3′-blocking moiety,“3-blocked” oligonucleotides are not necessarily terminatingoligonucleotides. Other oligonucleotides used herein, e.g., promoteroligonucleotides and capping oligonucleotides, are typically ornecessarily 3′-blocked as well.

As used herein, an “insertion sequence” is a sequence positioned betweenthe first region (i.e., template binding portion) and the second regionof a promoter oligonucleotide. Insertion sequences are preferably 5 to20 nucleotides in length, more preferably 6 to 18 nucleotides in length,and most preferably 6 to 12 nucleotides in length. The inclusion ofinsertion sequences in promoter oligonucleotides increases the rate atwhich RNA amplification products are formed.

“Target capture,” as used herein, includes any technique effective toremove all or substantially all unhybridized inactivatable targetcapture oligomer, as well as any other nucleic acid reagent, non-targetnucleic acid, unhybridized target nucleic acid or non nucleic acidcomponent in a reaction mixture. Generally, target capture involvescapturing a target polynucleotide onto a solid support, such asmagnetically attractable particles, where the solid support retains thetarget polynucleotide during one or more washing steps of the targetpolynucleotide purification procedure. In this way, a targetpolynucleotide is substantially purified from unhybridized inactivatabletarget capture oligomer, as well as any other nucleic acid reagent,non-target nucleic acid, unhybridized target nucleic acid or non nucleicacid component prior to any subsequent analysis steps. Numerous targetcapture methods are known and suitable for use in conjunction with themethods described herein.

For example, one illustrative approach described in U.S. Pub. No. US20060068417 A1 uses at least one capture probe oligonucleotide thatcontains a target-complementary region and a member of a specificbinding pair that joins a target nucleic acid to an immobilized probe ona capture support, thus forming a capture hybrid that is separated fromother sample components of a sample. Another illustrative method,Weisburg et al., U.S. Pat. No. 6,110,678, describes a method forcapturing a target polynucleotide in a sample onto a solid support, suchas magnetically attractable particles, with an attached immobilizedprobe by using a capture probe and two different hybridizationconditions, which preferably differ in temperature only. The twohybridization conditions control the order of hybridization, where thefirst hybridization conditions allow hybridization of the capture probeto the target polynucleotide, and the second hybridization conditionsallow hybridization of the capture probe to the immobilized probe. Themethod may be used to detect the presence of a target polynucleotide ina sample by detecting the captured target polynucleotide or amplifiedtarget polynucleotide. Another illustrative target capture techniqueinvolves a hybridization sandwich technique for capturing and fordetecting the presence of a target polynucleotide. See Ranki et al.,U.S. Pat. No. 4,486,539. The technique involves the capture of thetarget polynucleotide by a probe bound to a solid support andhybridization of a detection probe to the captured targetpolynucleotide. Detection probes not hybridized to a target nucleic acidare readily washed away from the solid support. Thus, remaining label isassociated with the target polynucleotide initially present in thesample.

Another illustrative target capture technique involves a method thatuses a mediator polynucleotide that hybridizes to both a target nucleicacid and to a polynucleotide fixed on a solid support. See Stabinsky,U.S. Pat. No. 4,751,177. The mediator polynucleotide joins the targetpolynucleotide to the solid support to produce a bound target. A labeledprobe can be hybridized to the bound target and unbound labeled pro canbe washed away from the solid support. Yet another illustrative targetcapture technique is disclosed by Englelhardt, U.S. Pat. No. 5,288,609,which describes a method for detecting a target polynucleotide. Themethod utilizes two single-stranded polynucleotide segmentscomplementary to the same or opposite strands of the target and resultsin the formation of a double hybrid with the target polynucleotide. Inone embodiment, the hybrid is captured onto a support.

In another illustrative target capture technique, methods and kits fordetecting nucleic acids use oligonucleotide primers labeled withspecific binding partners to immobilize primers and primer extensionproducts. See Burdick et al., European Pat. Appln. No. 0 370 694 A2. Thelabel specifically complexes with its receptor which is bound to a solidsupport. The above capture techniques are illustrative only, and notlimiting. Indeed, essentially any technique available to the skilledartisan may be used provided it is effective for removing all orsubstantially all unhybridized inactivatable target capture oligomer,after hybridization of inactivatable target capture oligomer with atarget nucleic acid sequence but prior to downstream analysis (e.g.,amplification) of the target nucleic acid sequence.

The term “inactivatable target capture oligomer” refers to anoligonucleotide that is capable of selectively hybridizing to a targetnucleic acid. Inactivatable target capture oligomers of the currentinvention preferably comprise a target hybridizing region, a tag-closingregion and a binding pair member. Two or more of these regions can bejoined as contiguous nucleic acid sequences. Alternatively, any tworegions can be joined using a non-nucleotide linker. Alternativelystill, the tag-closing sequence can be a discrete molecule. Contiguouslyjoined regions and members can be arranged 5′ to 3′ or 3′ to 5′,depending on preference. When non-nucleotide linkers are used to joinregions, orientations can further include 5′ to 5′ or 3′ to 3′.Preferably, the regions are joined as contiguous nucleic acid sequences,wherein the target hybridizing region is joined at its 3′ end to the 5′end of a tag-closing region and the tag-closing region is joined at its3′end to a binding pair member. If the binding pair member is a nucleicacid sequence region, then the tag-closing region is joined at its 3′end to the 5′ end of the binding pair member. In an alternativeorientation, the target hybridizing region is joined at its 5′ end tothe 3′ end of a tag-closing region and the tag-closing region is joinedat its 5′end to a binding pair member, which, if the binding pair memberis a nucleic acid sequence region, then the tag-closing region is joinedat its 5′ end to the 3′ end of the binding pair member. When the bindingpair member is a non-nucleic acid sequence, it can be covalently linkedto the nucleic acid tag-closing region, for example. (See e.g., FIGS.24-25) Compositions and methods for linking nucleic acid regions and forlinking non-nucleic acid moieties to a nucleic acid are known in theart. Other configurations of the inactivatable target capture oligomer,there is provided a target region joined on one of its ends to a bindingpair member and the tag-closing region is a discrete molecule or isjoined by a linker to an end on the target capture region that isopposite the end joined to the binding pair member (e.g., FIGS. 22-23).Essentially, the binding pair member and the tag-closing region arebound to opposites ends of the target hybridizing region.

Inactivatable target capture oligomers of the current invention areconfigured to provide, under certain conditions, an active configurationand an inactive configuration. Thus, the tag-closing region isconfigured so that under a first set of conditions wherein the targethybridizing region stably hybridizes with a target nucleic acidsequence, this tag-closing region does not stably hybridize with thetarget nucleic acid or any known non-target or contaminating nucleicacid sequences believed to be in a sample. But, under a second set ofconditions, the tag-closing region hybridizes with the targethybridizing region, thereby preventing further hybridization of theinactivated inactivatable target capture oligomer with any non-target orcontaminating nucleic acid sequences. Preferably, the active andinactive configurations are linear or hairpin configurations,respectively. However, in the embodiments wherein a tag-closing regionis a discrete molecule from the target hybridizing region and bindingpair member molecule, then active and inactive configurations for thesetwo discrete molecules occurs when the molecules are dissociated andhybridized, respectively. Preferably, the hairpin configuration formingthe inactive inactivatable target capture oligomer comprises aself-hybridization event wherein the tag-closing region and a portion ofthe target hybridizing region hybridize together thereby blocking thetarget hybridizing region from further hybridizing a nucleic acid in thereaction mixture. Alternatively for the configuration wherein thebinding pair member and the tag-closing region are joined to oppositeends of the target hybridizing region, the inactivated configuration isstill a hairpin configuration wherein the tag-closing region and aportion of the target hybridizing region hybridize together therebyblocking the target hybridizing region from further hybridizing anucleic acid in the reaction mixture. In configurations wherein thetag-closing region is a discreet oligomer, hybridization of the discreteclosing oligomer to the target hybridization region inactivatesinactivatable target capture oligomer.

Preferably the binding pair member of an inactivatable target captureoligomer is a substantially homopolymeric nucleic acid sequence that iscomplementary to a substantially homopolymeric nucleic acid sequencesecond binding pair member of a solid support. Thus, members of thebinding pair are complementary polynucleotides that are unlikely toparticipate in stable, non-specific binding reactions (e.g., poly(dA)and poly(dT) sequences of similar length). Weisburg et al., U.S. Pat.No. 6,534,273. Non-nucleic acid binding pair members include, but arenot limited to, ligand-ligate (e.g., avidin-biotin linkage). While thepreferred capture probe binds to both the target nucleic acid and asecond, immobilized binding pair member under the same conditions, thecapture probe may be designed so that the target hybridizing sequenceand the second binding pair member bind to their counterparts underdifferent conditions. In this way, the capture probe may be designed sothat it first binds the target nucleic acid under more favorable insolution kinetics before adjusting the conditions to permit binding ofthe first binding pair member to the second binding pair member.

The phrases “tag-closing sequence,” “capture probe closing sequence” and“capture probe closing oligonucleotide” refer to an oligonucleotidesequence that is complementary to a portion of a target hybridizingsequence of an inactivatable target capture oligomer. The length andsequence of the tag-closing sequence are selected so that thetag-closing sequence does not stably hybridize to the target hybridizingsequence of the inactivatable target capture oligomer under a first setof conditions permitting stable hybridization of the target hybridizingsequence to a target nucleic acid. The tag-closing sequence may includeabasic nucleotides or base mismatches with the target hybridizingsequence. Provided an inactivatable target capture oligomer is nothybridized to a target nucleic acid, the tag-closing sequence stablyhybridizes to the target hybridizing sequence under a second set of lessstringent conditions, thus “inactivating” or blocking the inactivatabletarget capture oligomer from hybridizing to the non-target andcontaminating nucleic acids (“closed capture probe”). As describedherein, the tag-closing sequence may be in the form of a discreteoligonucleotide or it may be joined to an end of the target hybridizingsequence. When joined to the target hybridizing sequence, it may bejoined as a contiguous nucleotide sequence or through a non-nucleotidelinker. Further, the tag-closing sequence may be joined to the targethybridizing sequence at an end of the target hybridizing sequenceopposite the end joined to the binding pair member. Alternatively, thetag-closing may be joined on one of its ends to the target hybridizingregion and on the other of its ends to the binding pair member. When thetag-closing sequence is not a discrete molecule, then the inactivatabletarget capture oligomer forms a hairpin structure under the second setof conditions. If the inactivatable target capture oligomer has aterminal 3′ end that can be used in a primer-based extension, and suchextension is not desired, then that 3′ terminal residue is preferablymodified to prevent the initiation of DNA synthesis therefrom, which caninclude using a blocking moiety situated at its 3′-terminus. In somealternative embodiments wherein the inactivatable target captureoligomer is also used as a priming oligomer, the inactivatable targetcapture oligomer can further comprise a tag region to introduce a uniquesite into a first amplification product. Subsequent amplification canuse an amplification oligomer that hybridizes to this tag region, or acomplement thereof. Optionally, the tag region can be the same as or canoverlap with the tag-closing region. If the inactivatable target captureoligomer forms a hairpin structure under the second set of conditions,then the target hybridizing sequence preferably hybridizes to thetag-closing sequence. The tag-closing sequence is at least 3 but no morethan about 20 bases in length, preferably at least 5 but no more than 20bases in length, and more preferably from 6 to 14 bases in length, andmore preferably still 6 to 9 nucleobases in length.

By “probe” or “detection probe” is meant a molecule comprising anoligonucleotide having a base sequence partly or completelycomplementary to a region of a target sequence sought to be detected, soas to hybridize thereto under stringent hybridization conditions. Aswould be understood by someone having ordinary skill in the art, a probecomprises an isolated nucleic acid molecule, or an analog thereof, in aform not found in nature without human intervention (e.g., recombinedwith foreign nucleic acid, isolated, or purified to some extent).Detection probes may have additional nucleosides or nucleobases outsideof the targeted region so long as such nucleosides or nucleobases do notsubstantially affect hybridization under stringent hybridizationconditions and, in the case of detection probes, do not preventpreferential hybridization to the target nucleic acid. Anon-complementary sequence may also be included, such as a targetcapture sequence (generally a homopolymer tract, such as a poly-A,poly-T or poly-U tail), promoter sequence, a binding site for RNAtranscription, a restriction endonuclease recognition site, or maycontain sequences which will confer a desired secondary or tertiarystructure, such as a catalytic active site or a hairpin structure on theprobe, on the target nucleic acid, or both. Detection probes preferablyinclude at least one detectable label. The label may be any suitablelabeling substance, including but not limited to a radioisotope, anenzyme, an enzyme cofactor, an enzyme substrate, a dye, a hapten, achemiluminescent molecule, a fluorescent molecule, a phosphorescentmolecule, an electrochemiluminescent molecule, a chromophore, a basesequence region that is unable to stably hybridize to the target nucleicacid under the stated conditions, and mixtures of these. In oneparticularly preferred embodiment, the label is an acridinium ester.Probes may also include interacting labels that emit different signals,depending on whether the probes have hybridized to target sequences.Examples of interacting labels include enzyme/substrates,enzyme/cofactor, luminescent/quencher, luminescent/adduct, dye dimers,and Förrester energy transfer pairs. Certain probes do not include alabel; for example, non-labeled capture probes may be used to enrich fortarget sequences or replicates thereof, which may then be detected by asecond “detection” probe. See, e.g., Weisburg et al., U.S. Pat. No.6,534,273. While detection probes are typically labeled, certaindetection technologies do not require that the probe be labeled. See,e.g., Nygren et al., U.S. Pat. No. 6,060,237. Probes of a definedsequence may be produced by techniques known to those of ordinary skillin the art, such as by chemical synthesis, and by in vitro or in vivoexpression from recombinant nucleic acid molecules. Preferably probesare 10 to 100 nucleotides in length, more preferably 12 to 50 bases inlength, and even more preferably 18 to 35 bases in length.

By “stable” or “stable for detection” is meant that the temperature of areaction mixture is at least 2.deg.C below the melting temperature of anucleic acid duplex. The temperature of the reaction mixture is morepreferably at least 5.deg.C below the melting temperature of the nucleicacid duplex, and even more preferably at least 10.deg.C below themelting temperature of the reaction mixture.

“Nucleic Acid Identity” means a nucleic acid comprising a contiguousbase region that is at least 80%, 90%, or 100% identical to a contiguousbase region of a reference nucleic acid. For short nucleic acids, e.g.,certain oligonucleotides of the present invention, the degree ofidentity between a base region of a “query” nucleic acid and a baseregion of a reference nucleic acid can be determined by manualalignment. “Identity” is determined by comparing just the sequence ofnitrogenous bases, irrespective of the sugar and backbone regions of thenucleic acids being compared. Thus, the query:reference base sequencealignment may be DNA:DNA, RNA:RNA, DNA:RNA, RNA:DNA, or any combinationsor analogs thereof. Equivalent RNA and DNA base sequences can becompared by converting U's (in RNA) to T's (in DNA). Differences betweentwo nucleic acid sequences being compared can include nucleotideadditions, deletions, substitutions and modification in one sequencecompared to the other.

A “template” is a nucleic acid molecule that is being copied by anucleic acid polymerase. A template may be single-stranded,double-stranded or partially double-stranded, depending on thepolymerase. The synthesized copy is complementary to the template or toat least one strand of a double-stranded or partially double-strandedtemplate. Both RNA and DNA are typically synthesized in the 5′-to-3′direction and the two strands of a nucleic acid duplex are aligned sothat the 5′-termini of the two strands are at opposite ends of theduplex (and, by necessity, so then are the 3′-termini). Templatescomprise the target sequence, and in some instances also comprisesecondary primer extension products, such as tag sequences or promotersequences.

A “DNA-dependent DNA polymerase” is an enzyme that synthesizes acomplementary DNA copy from a DNA template. Examples are Taq DNApolymerase, a highly thermostable DNA polymerase from the thermophilicbacterium Thermus aquaticus, for PCR amplification reactions, DNApolymerase I from E. coli, bacteriophage T7 DNA polymerase, or DNApolymerases from bacteriophages T4, Phi-29, M2, or T5. DNA-dependent DNApolymerases may be the naturally occurring enzymes isolated frombacteria or bacteriophages or expressed recombinantly, or may bemodified or “evolved” forms which have been engineered to possesscertain desirable characteristics, e.g., thermostability, or the abilityto recognize or synthesize a DNA strand from various modified templates.All known DNA-dependent DNA polymerases require a complementary primerto initiate synthesis. It is known that under suitable conditions aDNA-dependent DNA polymerase may synthesize a complementary DNA copyfrom an RNA template. RNA-dependent DNA polymerases (described below)typically also have DNA-dependent DNA polymerase activity. An example ofsuch a polymerase is the MasterAmp.sup.™ Tth DNA Polymerase, which hasboth DNA-dependent and RNA-dependent (i.e., reverse transcriptase) DNApolymerase activities that can be used in both PCR and RT-PCRamplification reactions (Epicentre Biotechnologies, Madison, Wis.).

A “DNA-dependent RNA polymerase” or “transcriptase” is an enzyme thatsynthesizes multiple RNA copies from a double-stranded orpartially-double-stranded DNA molecule having a promoter sequence thatis usually double-stranded. The RNA molecules (“transcripts”) aresynthesized in the 5′-to-3′ direction beginning at a specific positionjust downstream of the promoter. Examples of transcriptases are theDNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6.

An “RNA-dependent DNA polymerase” or “reverse transcriptase” (“RT”) isan enzyme that synthesizes a complementary DNA copy from an RNAtemplate. All known reverse transcriptases also have the ability to makea complementary DNA copy from a DNA template; thus, they are both RNA-and DNA-dependent DNA polymerases. RTs may also have an RNAse Hactivity. Preferred is reverse transcriptase derived from Maloney murineleukemia virus (MMLV-RT). A primer is required to initiate synthesiswith both RNA and DNA templates.

As used herein, a “selective RNAse” is an enzyme that degrades the RNAportion of an RNA:DNA duplex but not single-stranded RNA,double-stranded RNA or DNA. An exemplary selective RNAse is RNAse H.Enzymes other than RNAse H but that possess the same or similar activityare also contemplated herein. Selective RNAses may be endonucleases orexonucleases. Most reverse transcriptase enzymes contain an RNAse Hactivity in addition to their polymerase activities. However, othersources of the RNAse H are available without an associated polymeraseactivity. The degradation may result in separation of RNA from a RNA:DNAcomplex. Alternatively, a selective RNAse may simply cut the RNA atvarious locations such that portions of the RNA melt off or permitenzymes to unwind portions of the RNA. Other enzymes that selectivelydegrade RNA target sequences or RNA products of the present inventionare readily apparent to those of ordinary skill in the art.

Sense/Antisense Strand(s): Discussions of nucleic acid synthesis aregreatly simplified and clarified by adopting terms to name the twocomplementary strands of a nucleic acid duplex. Traditionally, thestrand encoding the sequences used to produce proteins or structuralRNAs are designated as the “sense (+)” strand and its complement the“antisense (−)” strand. It is now known that in many cases, both strandsare functional, and so the assignment of the designation “sense” to oneand “antisense” to the other has become arbitrary. Nevertheless, theterms can be helpful for designating the sequence orientation of nucleicacids and may be employed herein for that purpose.

The term “specificity,” in the context of a selective hybridization andcapture system, or in the context of an amplification system, is usedherein to refer to the characteristic that describes it's the ability ofa system to distinguish between target and non-target sequences,dependent upon sequence and assay conditions. In terms of selectivetarget nucleic hybridization and capture, specificity generally refersto the ratio of the number of target nucleic acids hybridized andcaptured to the number of non-target and contaminating nucleic acidshybridized and captures, as discussed herein. In terms of nucleic acidamplification, specificity generally refers to the ratio of the numberof specific amplicons produced to the number of side-products (i.e., thesignal-to-noise ratio), as discussed herein.

The term “sensitivity” is used herein to refer to the precision withwhich a nucleic acid amplification reaction can be detected orquantitated. The sensitivity of an amplification reaction is generally ameasure of the smallest copy number of the target nucleic acid that canbe reliably detected in the amplification system, and often depends, forexample, on the detection assay being employed, and the specificity ofthe capture and amplification reactions, e.g., side-product ornon-target/contaminant amplification reactions.

A “bioprocess,” as used herein, refers generally to any process in whichliving cells, or components thereof, are present, either intended orunintended. For example, essentially any manufacturing or other processthat employs one or more samples or sample streams, at least one ofwhich comprises living cells, or components thereof, or may comprisesuch cells or components as a result of unintended contamination, isconsidered a bioprocess. In many such processes it is desirable to havethe ability to detect, identify and/or control the presence and/orsources of living cells or components thereof within a process. Usingthe compositions, kits and methods of the present invention, forexample, the presence and/or sources of target nucleic acids from knownor suspected bioprocess-contaminating microorganisms or other biologicalmaterial or components thereof in one or more bioprocess samples orstreams may be monitored. In addition, the purification/sterilizationrequirements within certain samples/streams of a bioprocess may beadvantageously reduced using the methods of the invention as set forthherein.

As discussed above, the present invention is directed generally tocompositions, kits and methods for the selective hybridization of aninactivatable target capture oligomer to a target nucleic acid. Theactive form of the inactivatable target capture oligomer selectivelyhybridizes to target nucleic acids under a first set of conditions.Unhybridized inactivatable target capture oligomers are inactivatedunder a second set of conditions. In activating the inactivatable targetcapture oligomer substantially reduces or eliminates non-specificbinding of the capture probe to any non-target nucleic acids orcontaminating nucleic acids present in a reaction mixture. Furtherprovided, hybridization complexes comprising inactivatable targetcapture oligomers and target nucleic acids can be captured, for exampleby providing a solid support comprising a complementary second bindingpair member. Selectively hybridized nucleic acids are then available fordownstream analyses, such as nucleic acid amplification. Non-targetnucleic acids and/or contaminating nucleic acids are, thusly,substantially reduced or eliminated, thereby providing for improvementsin subsequent analyses. For example, when the downstream analyses isamplification and detection of a target nucleic acid, there is areduction or elimination in false positive amplification signalsresulting from contaminating nucleic acid material that may be presentin one or more reagents, samples or components that are used in anamplification reaction, or that may be present in the environment inwhich amplification reactions are performed. The invention furtheroffers the advantage of requiring less stringent purification and/orsterility efforts conventionally needed in order to ensure that enzymes,other reagents, labwares, and reaction environments used in subsequentanalyses reactions, are free of bacterial and other nucleic acidcontamination that may yield false positive results. Accordingly, themethods of the invention are particularly useful in detecting,monitoring and/or quantitating target nucleic acids from a variety ofsamples, including those typically known to encounter diminished resultsdue to non-target nucleic acids and/or contaminating nucleic acids. Theamplification reactions can be essentially any amplification procedurerequiring a template-binding priming oligonucleotide capable ofextension in the presence of nucleic acid polymerase.

FIGS. 26 and 27 are an exemplary illustration of selective hybridization(FIG. 26) and capture (FIG. 27) of a target nucleic acid. Furtherillustrated in FIG. 27 is an optional wash step. Also illustrated andoptional in these FIGS. is the inclusion of a heterologous amplificationoligomer and a terminating oligomer. The exemplary illustration of theseFIGS. 26-27, then, is for a downstream amplification reaction,including, but not being limited to, those illustrated in FIGS. 1, 3A-B,5, 7 and 9. The compositions, kits and methods of the current inventionare not limited to hybridizing and capturing target nucleic acids for adownstream amplification reaction.

In FIG. 26, step A illustrates an inactivatable target capture oligomercomprising a target hybridizing region, a tag-closing region and abinding pair member. The inactivatable target capture oligomer isillustrated in the closed configuration. Also illustrated in step A is aprimer, a terminating oligomer and a target nucleic acid. Preferably,though not necessarily, the primer member comprises a tag sequence (notshown), and so it a heterologous amplification oligomer. Thedesignations “m” and “n” represent copy numbers for these components andthe target nucleic acid, respectively. A first conditions in thenachieved so that in step B the inactivatable target capture oligomer isin an active configuration. This first condition is sufficientlystringent that the active inactivatable target capture oligomer, theprimer and the terminating oligomer hybridize to the target nucleicacid, forming a preannealing complex. Step C. The number of preannealingcomplexes formed in a reaction will be limited because of the lesser ofthe added components, the target and/or other limiting factors. Thus,the number of complexes formed is represented by the “o” designation.Therefore, step C indicates that unhybridized active inactivatabletarget capture oligomer, primer and terminating oligomer are present inan amount of m-o, while excess target nucleic acids are present in anamount of n-o, wherein if o>m the amount is 0 and if o>n the amount is0. Ordinarily skilled artisans understand that such a reaction can haveexcess components or target. A second set of conditions is achieved nextto inactivate any unhybridized inactivatable target capture oligomers,thereby substantially reducing or eliminating their ability to hybridizeto non-target nucleic acids or contaminating nucleic acids; step D. In areaction that includes a primer oligomer, and wherein that primeroligomer is a heterologous amplification oligomer, a similarinactivating event can take place under these second conditions.

FIG. 27 illustrates the addition of a solid support comprising acomplementary second binding pair member; step A. Ordinarily skilledartisans understand capture of nucleic acids using solid supportscomprising second binding pair members. In a preferred embodiment, thesolid support is a magnetic bead comprising a substantiallyhomopolymeric binding pair member that is complementary to the bindingpair member of the inactivatable target capture oligomer. At step B thepreannealing complex formed in step D of FIG. 26 is further complexedwith the solid support comprising a second binding pair member.Preferably at this point, the inactive inactivatable target captureoligomer, the unhybridized primer and terminating oligomers and anyunhybridized target nucleic acids are removed from the captured complexin a series of wash and rinse steps. Further, any non-target orcontaminating nucleic acids in the reaction mixture are removed from thereaction by the wash/rinse steps. Thus, at C there is illustrated acaptured complex that is isolated away from the inactivatedinactivatable target capture oligomer, the unhybridized primer andterminating oligomers and any unhybridized target nucleic acids.Captured target nucleic acids are then used in a downstream procedure.In the example illustrated in FIGS. 26 and 27, the captured targetnucleic acid comprises components for an amplification reaction. Thus,the illustrated captured target nucleic acids can be eluted to, forexample, step 1—of any one of FIG. 1, 3A, 5, 7 or 9. Or, alternatively,a polymerase can be added at one of the steps in FIG. 27 to synthesize acomplementary copy of the target nucleic acid, thereby providing anucleic acid strand that is complementary to the target and thatincorporates the sequence of the primer member. In the embodimentswherein that primer member is a heterologous amplification oligomer,then a tag sequence is further incorporated into the nascent sequence.In these instances, the captured target nucleic acids and complementarystrand containing a tag sequence can be eluted into, for example, step 3of FIG. 1, step 3 of FIG. 3A, step 5 of FIG. 5, step 5 of FIG. 7, orstep 3 of FIG. 9. Such are merely exemplary and do not limit theinvention.

Ordinarily skilled artisans in possession of this specification willunderstand that the compositions, kits and methods of the currentinvention are not limited to downstream analyses that are amplificationreactions. And further, when the downstream analyses are amplificationreactions, the types of reactions and the order of the steps asillustrated and exemplified herein, do not limit the invention. Thefollowing further illustrates some of the exemplary downstreamamplification reactions.

FIG. 1 illustrates an adaptation of an isothermal, transcription-basedamplification reaction known as reverse transcription-mediatedamplification (rTMA), various aspects of which are disclosed in Beckeret al., U.S. Pub. No. U.S. 2006-0046265 A1. The reaction of thisillustrative embodiment is initiated by treating an RNA target sequencein a nucleic acid sample with both a tagged priming oligonucleotide(also referred to as a heterologous amplification oligomer) and aterminating oligonucleotide. The tagged priming oligonucleotide includesa target hybridizing sequence that hybridizes to a 3′-end of the targetsequence and a tag sequence (also referred to as a tags sequence)situated 5′ to the target hybridizing sequence. The terminatingoligonucleotide hybridizes to a target nucleic acid containing thetarget sequence in the vicinity of the 5′-end of the target sequence.The terminating oligonucleotide is used to end primer extension of anascent nucleic acid that includes the tagged priming oligonucleotide.Thus, the target nucleic acid forms a stable complex with the taggedpriming oligonucleotide at the 3′-end of the target sequence and theterminating oligonucleotide located adjacent to or near the 5′-end ofthe target sequence prior to initiating a primer extension reaction. SeeFIG. 1, Step 1. Unhybridized tagged priming oligonucleotide ispreferably made unavailable for hybridization to the target sequenceprior to initiating a primer extension reaction with the tagged primingoligonucleotide, preferably by inactivating and/or removing theunhybridized tagged priming oligonucleotide from the nucleic acidsample. An extension reaction is then initiated from the 3′-end of thehybridized tagged priming oligonucleotide with a DNA polymerase, e.g.,reverse transcriptase, to produce a first DNA primer extension productthat includes the tag sequence and a region complementary to the targetsequence. See FIG. 1, Steps 2 and 3. The first DNA primer extensionproduct is then separated from the target sequence using an enzyme thatselectively degrades the target sequence (e.g., RNAse H activity). SeeFIG. 1, Step 4.

Next, the first DNA primer extension product is treated with a promoteroligonucleotide having a hybridizing sequence and a promoter for an RNApolymerase situated 5′ to the hybridizing sequence. The hybridizingsequence hybridizes to a region of the first DNA primer extensionproduct that is complementary to the 3′-end of the target sequence,thereby forming a promoter oligonucleotide:first DNA primer extensionproduct hybrid. In the illustrated reaction, the promoteroligonucleotide is modified to prevent the initiation of DNA synthesis,preferably by situating a blocking moiety at the 3′-end of the promoteroligonucleotide (e.g., nucleotide sequence having a 3′-to-5′orientation). See FIG. 1, Step 5. The 3′-end of the first DNA primerextension product is preferably extended to add a sequence complementaryto the promoter, resulting in the formation of a double-strandedpromoter sequence. See FIG. 1, Steps 6 and 7. Multiple copies of a firstRNA product complementary to at least a portion of the first DNA primerextension product, not including the promoter portion, are thentranscribed using an RNA polymerase which recognizes the double-strandedpromoter and initiates transcription therefrom. See FIG. 1, Steps 8 and9. As a result, the base sequence of the first RNA product issubstantially identical to the base sequence of the target sequence andthe complement of the tag sequence provided by the heterologousamplification oligomer in steps 1-3.

In amplification reactions that use a heterologous amplificationoligomer, subsequent rounds of amplification can take advantage of thisincorporated tag sequence by including an amplification oligomer thathybridizes to the tag sequence. Because the tag sequence is a uniquenucleic acid sequence relative to a reaction mix's nucleic acidenvironment, which includes the target nucleic acid sequence, andnucleic acids sequences of suspected non-target nucleic acids andcontaminating nucleic acids, subsequent amplification rounds aresubstantially limited to or exclusive for the target nucleic acidsequence. In FIG. 1, first RNA products are treated with a primingoligonucleotide that hybridizes to the complement of the tag sequence toform a priming oligonucleotide:first RNA product hybrid, and the 3′-endof the priming oligonucleotide is extended with the DNA polymerase toproduce a second DNA primer extension product complementary to the firstRNA product. See FIG. 1, Steps 10-12. The second DNA primer extensionproduct is then separated from the first RNA product using an enzymethat selectively degrades the first RNAproduct (e.g., RNAse H activity).See FIG. 1, Step 13.

The second DNA primer extension product is treated with the promoteroligonucleotide, which hybridizes to the 3′-end of the second DNA primerextension product to form a promoter oligonucleotide:second DNA primerextension product hybrid. See FIG. 1, Step 14. The promoteroligonucleotide:second DNA primer extension product hybrid thenre-enters the amplification cycle at Step 6 of FIG. 1, wheretranscription is initiated from the double-stranded promoter and thecycle continues.

FIGS. 3A-B illustrates an adaptation of an isothermal,transcription-based amplification reaction referred to astranscription-mediated amplification (TMA), various aspects of which aredisclosed in Kacian et al., U.S. Pat. Nos. 5,399,491 and 5,824,518. Thereaction of this illustrative embodiment is initiated by treating an RNAtarget sequence in a nucleic acid sample with a heterologousamplification oligomer comprising a promoter region (i.e., a taggedpromoter oligonucleotide). Thus, the tagged promoter oligonucleotideincludes a tag sequence, a target hybridizing sequence and a promotersequence for an RNA polymerase, where the target hybridizing sequencehybridizes to a 3′-end of the target sequence. The target sequence formsa stable complex with the tagged promoter oligonucleotide at the 3′-endof the target sequence prior to initiating a primer extension reaction.See FIG. 3A, Step 1. The promoter sequence is situated 5′ to the tagsequence, and the tag sequence is situated 5′ to the target hybridizingsequence. Unhybridized tagged promoter oligonucleotide is preferablymade unavailable for hybridization to the target sequence prior toinitiating a primer extension reaction with the tagged primingoligonucleotide, preferably by inactivating and/or removing theunhybridized tagged priming oligonucleotide from the nucleic acidsample. An extension reaction is then initiated from the 3′-end of thetagged promoter oligonucleotide with a DNA polymerase, e.g., reversetranscriptase, to produce a first DNA primer extension product thatincludes the tag and promoter sequence and a region complementary to thetarget sequence. See FIG. 1, Steps 2 and 3. The first DNA primerextension product is then separated from the target sequence to which itis hybridized using an enzyme that selectively degrades that portion ofthe target sequence that is hybridized to the first DNA primer extensionproduct (e.g., RNAse H activity). See FIG. 3A, Step 4.

Next, the first DNA primer extension product is treated with a primingoligonucleotide which hybridizes to a region of the first DNA primerextension product that is complementary to a 5′-end of the targetsequence, thereby forming a priming oligonucleotide:first DNA primerextension product hybrid. See FIG. 3A, Step 5. The 3′-end of the primingoligonucleotide is extended by a DNA polymerase to produce a second DNAprimer extension product complementary to at least a portion of thefirst DNA primer extension product, and containing a double-strandedpromoter sequence. See FIG. 3A, Steps 6 and 7. This second DNA primerextension product is used as a template to transcribe multiple copies ofa first RNA product complementary to the second DNA primer extensionproduct, not including the promoter portion, using an RNA polymerasethat recognizes the double-stranded promoter and initiates transcriptiontherefrom. See FIGS. 3A-B, Step 8 and 9. The base sequence of the firstRNA product is substantially identical to the base sequence of thecomplement of the target sequence and the tag sequence, therebyproviding the tag sequence for a subsequent amplification oligomer tobind.

The first RNA product is treated with the priming oligonucleotide, the3′-end of which is extended by the DNA polymerase to produce a third DNAprimer extension product complementary to the first RNA product. SeeFIG. 3B, Steps 10-12. The third DNA primer extension product is thenseparated from the first RNA product using an enzyme that selectivelydegrades the first RNA product (e.g., RNAse H activity). See FIG. 3B,Step 13. The third DNA primer extension product is treated with apromoter oligonucleotide having a hybridizing sequence which hybridizesto a complement of the tag sequence at the 3′-end of the third DNAprimer extension product, and further comprises a promoter for an RNApolymerase which is situated 5′ to the hybridizing sequence. See FIG.3B, Step 14. The 3′-end of the third DNA primer extension product isextended to add sequence complementary to the promoter sequence. SeeFIG. 3B, Step 15. The 3′-end of the promoter oligonucleotide is extendedwith the DNA polymerase to produce a fourth DNA primer extension productcomplementary to the third DNA primer extension product. See FIG. 3B,Step 16. Multiple copies of a second RNA product complementary to thethird DNA primer extension product, not including the promoter portion,are transcribed from the double-stranded promoter and re-enter theamplification cycle at Step 9 of FIG. 3B. The base sequence of thesecond RNA product is substantially identical to the base sequence ofthe tag sequence and complement of the target sequence.

FIG. 5 illustrates an adaptation of an rTMA amplification reaction foramplifying a DNA target sequence, various aspects of which are disclosedin Becker et al., U.S. Pub. No. U.S. 2007-0202523 A1. The reaction ofthis illustrative embodiment is initiated by treating a DNA targetsequence in a nucleic acid sample with a tagged priming oligonucleotideand a terminating oligonucleotide. The tagged priming oligonucleotideincludes a target hybridizing sequence hybridized to a 3′-end of thetarget sequence and a tag sequence situated 5′ to the target hybridizingsequence. The target hybridizing sequence preferably hybridizes to asingle-stranded form of the target sequence, although it may hybridizeto a double-stranded form of the target sequence through strandinvasion, which can be facilitated by, for example, DNA breathing (e.g.,AT rich regions), low salt conditions, and/or the use of DMSO and/orosmolytes, such as betaine. The target sequence is preferably renderedsingle-stranded by heating the nucleic acid sample. The terminatingoligonucleotide hybridizes to a region of a target nucleic acidcontaining the target sequence in the vicinity of the 5′-end of thetarget sequence. The terminating oligonucleotide is used to end primerextension of a nascent nucleic acid that includes the tagged primingoligonucleotide. Thus, the target nucleic acid forms a stable complexwith the tagged priming oligonucleotide at the 3′-end of the targetsequence and the terminating oligonucleotide located adjacent to or nearthe 5′-end of the target sequence. See FIG. 5, Steps 1-3. Unhybridizedtagged priming oligonucleotide is made unavailable for hybridization tothe target sequence prior to initiating a primer extension reaction withthe tagged priming oligonucleotide. An extension reaction is theninitiated from the 3′-end of the tagged priming oligonucleotide with aDNA polymerase, e.g., reverse transcriptase, to produce a first DNAprimer extension product that includes the tag sequence and a regioncomplementary to the target sequence. See FIG. 5, Steps 4 and 5.

The nucleic acid sample is further treated with a displaceroligonucleotide that hybridizes to the target nucleic acid upstream fromthe tagged oligonucleotide such that a primer extension reaction can beinitiated therefrom, so that the first DNA primer extension product isdisplaced when the DNA polymerase extends a 3′-end of the displaceroligonucleotide. See FIG. 5, Steps 6-8. The order of the illustratedsteps is not meant to imply that the nucleic acid sample of thisembodiment must be treated with the tagged priming oligonucleotidebefore it is treated with the displacer oligonucleotide to beoperational. In certain embodiments, it is preferable to have these twooligonucleotides hybridize to the target nucleic acid substantiallysimultaneously.

Next, the first DNA primer extension product is treated with a promoteroligonucleotide having a hybridizing sequence and a promoter for an RNApolymerase situated 5′ to the hybridizing sequence. The hybridizingsequence hybridizes to a region of the first DNA primer extensionproduct that is complementary to the 3′-end of the target sequence,thereby forming a promoter oligonucleotide:first DNA primer extensionproduct hybrid. In the illustrated reaction, the promoteroligonucleotide is modified to prevent the initiation of DNA synthesisby situating a blocking moiety at the 3′-end of the promoteroligonucleotide (e.g., nucleotide sequence having a 3′-to-5′orientation). See FIG. 5, Step 9. The 3′-end of the first DNA primerextension product is extended to add sequences complementary to thepromoter, resulting in the formation of a double-stranded promotersequence. See FIG. 5, Steps 10 and 11. Multiple copies of a first RNAproduct complementary to at least a portion of the first DNA primerextension product, not including the promoter, are transcribed using anRNA polymerase that recognizes the double-stranded promoter andinitiates transcription therefrom. See FIG. 5, steps 12 and 13. As aresult, the base sequence of the first RNA product is substantiallyidentical to the target sequence and the complement tag sequence.

The first RNA products are contacted with a priming oligonucleotide thathybridizes to the complement of the tag sequence to form a primingoligonucleotide:first RNA product hybrid, and the 3′-end of the primingoligonucleotide is extended with the DNA polymerase to produce a secondDNA primer extension product complementary to the first RNA product. SeeFIG. 5, steps 14-16. The second DNA primer extension product isseparated from the first RNA product using and enzyme that selectivelydegrades the first RNA product (e.g., RNAse H activity). See FIG. 5,Step 17. The second DNA primer extension product is treated with thepromoter oligonucleotide to form a promoter oligonucleotide:second DNAprimer extension product hybrid. See FIG. 5, Step 18. The promoteroligonucleotide:second primer extension product hybrid then re-entersthe amplification cycle at Step 10 of FIG. 5, where transcription isinitiated from the double-stranded promoter and the cycle continues.

FIG. 7 illustrates an adaptation of a PCR. The reaction of thisillustrative embodiment is initiated by treating a denatured DNA targetsequence in a nucleic acid sample with a tagged priming oligonucleotide.The tagged priming oligonucleotide includes a target hybridizingsequence that hybridizes to a 3′-end of the target sequence and a tagsequence situated 5′ to the target hybridizing sequence. Thus, thetarget sequence forms a stable complex with the tagged primingoligonucleotide at the 3′-end of the target sequence prior to initiatinga primer extension reaction. See FIG. 7, Steps 1-3. Unhybridized taggedpriming oligonucleotide is then made unavailable for hybridization tothe target sequence. An extension reaction is initiated from the 3′-endof the tagged priming oligonucleotide with a DNA polymerase, e.g., TaqDNA polymerase, to produce a first DNA primer extension product thatincludes the tag sequence and a region complementary to the targetsequence. See FIG. 7, Steps 4 and 5. Next, the double-stranded productresulting from the first primer extension reaction is denatured and thefirst DNA primer extension product is contacted with a first primingoligonucleotide that hybridizes to a region of the first DNA primerextension product that is complementary to the 5′-end of the targetsequence. See FIG. 7, Steps 6 and 7.

In a second primer extension reaction, the 3′-end of the first primingoligonucleotide is extended with the DNA polymerase to produce a secondDNA primer extension product that is complementary to a portion of thefirst primer extension product and includes the target sequence and thecomplement of the tag sequence. See FIG. 7, Steps 8 and 9. Thedouble-stranded product resulting from the second primer extensionreaction is denatured and the second DNA primer extension product iscontacted with a second priming oligonucleotide that hybridizes to thecomplement of the tag sequence. See FIG. 7, Steps 10 and 11. The 3′-endof the second priming oligonucleotide is then extended in a third primerextension reaction with the DNA polymerase to produce a third DNA primerextension product that is complementary to the second DNA primerextension product. FIG. 7, Steps 12 and 13. The double-stranded productresulting from the third primer extension reaction is denatured and thesecond and third DNA primer extension products are available forparticipation in the repeated cycles of a polymerase chain reactionusing as primers the first and second priming oligonucleotides. See FIG.7, Steps 14-16.

FIG. 9 illustrates an adaptation of RT-PCR. The reaction of thisillustrative embodiment is initiated by treating an RNA target sequencein a nucleic acid sample with a tagged priming oligonucleotide. Thetagged priming oligonucleotide includes a target hybridizing sequenceand a tag sequence situated 5′ to the target hybridizing sequence. Thus,the target sequence forms a stable complex with the tagged primingoligonucleotide at the 3′-end of the target sequence prior to initiatinga primer extension reaction. See FIG. 9, Step 1. Unhybridized taggedpriming oligonucleotide is made unavailable for hybridization to thetarget sequence. An extension reaction is then initiated from the 3′-endof the tagged priming oligonucleotide with a DNA polymerase, e.g.,MasterAmp.sup.™ Tth DNA Polymerase, to produce a first DNA primerextension product that includes the tag sequence and a regioncomplementary to the target sequence. See FIG. 9, Steps 2 and 3. Thefirst DNA primer extension product is then separated from the targetnucleic acid sequence to which it is hybridized using an enzyme thatselectively degrades that portion of a target nucleic acid containingthe target sequence that is complementary to the first DNA primerextension product (e.g., RNAse H activity). See FIG. 9, Step 4. Next,the first DNA primer extension product is treated with a first primingoligonucleotide that hybridizes to a region of the first DNA primerextension product that is complementary to the 5′-end of the targetsequence to form a first DNA primer extension product:first primingoligonucleotide hybrid. See FIG. 9, Step 5. A second primer extensionreaction extends the 3′-end of the first priming oligonucleotide withthe DNA polymerase to produce a DNA second primer extension productcomplementary to at least a portion of the first primer extensionproduct and includes the target sequence and the complement of the tagsequence. See FIG. 9, Steps 6 and 7. The first and second DNA primerextension products are then separated from each other by denaturation.See FIG. 9, Step 8. The first and second extension products are thenavailable to participate in the repeated cycles of a polymerase chainreaction using as primers the first priming oligonucleotide and a secondpriming oligonucleotide which hybridizes to the complement of the tagsequence. See FIG. 9, Steps 9 and 10; FIG. 7, Steps 13-16.

Preferably for these amplification reactions, heterologous amplificationoligomers that have not formed part of a tagged target nucleic acidsequence are inactivated prior to exposing the tagged target nucleicacid sequence to reagents and conditions sufficient for detectableamplification of a target nucleic acid sequence.

In addition to the methods described herein, the present invention isdrawn to kits comprising one or more of the reagents required forcarrying out the methods of the present invention. Kits comprisingvarious components used in carrying out the present invention may beconfigured for use in any procedure requiring amplification of nucleicacid target molecules, and such kits can be customized for variousdifferent end-users. Suitable kits may be prepared, for example, formicrobiological analysis, blood screening, disease diagnosis, watertesting, product release or sterility testing, environmental orindustrial analysis, food or beverage testing, or for general laboratoryuse. Kits of the present invention provide one or more of the componentsnecessary to carry out nucleic acid amplifications according to theinvention. Kits may include reagents suitable for amplifying nucleicacids from one particular target or may include reagents suitable foramplifying multiple targets. Kits of the present invention may furtherprovide reagents for real-time detection of one or more nucleic acidtargets in a single sample, for example, one or more self-hybridizingprobes as described above. Kits may comprise a carrier that may becompartmentalized to receive in close confinement one or more containerssuch as vials, test tubes, wells, and the like. Preferably at least oneof such containers contains one or more components or a mixture ofcomponents needed to perform the amplification methods of the presentinvention.

A kit according to one embodiment of the present invention can include,for example, in one or more containers, an inactivatable target captureoligomer of the current invention. Optionally, a kit may furthercomprise a heterologous amplification oligomer, a binding molecule orother means for terminating a primer extension reaction, and,optionally, an extender oligonucleotide and/or a cappingoligonucleotide. If real-time detection is used, the one or morecontainers may include one or more reagents for real-time detection ofat least one nucleic acid target sequence in a single sample, forexample, one or more self-hybridizing probes as described above. Anothercontainer may contain an enzyme reagent, such as a heat stable DNApolymerase for performing a PCR or RT-PCR reaction, or a mixture of areverse transcriptase (either with or without RNAse H activity), an RNApolymerase, and optionally an additional selective RNAse enzyme for atranscription-based amplification reaction. These enzymes may beprovided in concentrated form or at working concentration, usually in aform that promotes enzyme stability. The enzyme reagent may also beprovided in a lyophilized form. See Shen et al., “Stabilized EnzymeCompositions for Nucleic Acid Amplification,” U.S. Pat. No. 5,834,254.Another one or more containers may contain an amplification reagent inconcentrated form, e.g., 10×, 50×, or 100×, or at working concentration.An amplification reagent will contain one or more of the componentsnecessary to run the amplification reaction, e.g., a buffer, MgCl.sub.2,KCl, dNTPs, rNTPs, EDTA, stabilizing agents, etc. Certain of thecomponents, e.g., MgCl.sub.2 and rNTPs, may be provided separately fromthe remaining components, allowing the end user to titrate thesereagents to achieve more optimized amplification reactions. Another oneor more containers may include reagents for detection of amplificationproducts, including one or more labeled oligonucleotide probes. Probesmay be labeled in a number of alternative ways, e.g., with radioactiveisotopes, fluorescent labels, chemiluminescent labels, nuclear tags,bioluminescent labels, intercalating dyes, or enzyme labels. In someembodiments, a kit of the present invention will also include one ormore containers containing one or more positive and negative controltarget nucleic acids which can be utilized in amplification experimentsin order to validate the test amplifications carried out by the enduser. In some instances, one or more of the reagents listed above may becombined with an internal control. Of course, it is also possible tocombine one or more of these reagents in a single tube or othercontainers. Supports suitable for use with the invention, e.g., testtubes, multi-tube units, multi-well plates, etc., may also be suppliedwith kits of the invention. Finally a kit of the present invention mayinclude one or more instruction manuals.

Kits of the invention may contain virtually any combination of thecomponents set out above or described elsewhere herein. As one skilledin the art would recognize, the components supplied with kits of theinvention will vary with the intended use for the kits, and the intendedend user. Thus, kits may be specifically designed to perform variousfunctions set out in this application and the components of such kitswill vary accordingly.

The present invention is further drawn to various oligonucleotidesincluding, for example, the target specific oligonucleotides exemplifiedbelow. It is to be understood that oligonucleotides of the presentinvention may be DNA, RNA, DNA:RNA chimerics and analogs thereof, and,in any case, the present invention includes RNA equivalents of DNAoligonucleotides and DNA equivalents of RNA oligonucleotides.

Detection probes of the present invention may include, for example, anacridinium ester label, or labeled, self-hybridizing regions flankingthe sequence that hybridizes to the target sequence. In variousembodiments, these labeled oligonucleotide probes optionally orpreferably are synthesized to include at least one modified nucleotide,e.g., a 2′-O-ME ribonucleotide; or these labeled oligonucleotide probesoptionally or preferably are synthesized entirely of modifiednucleotides, e.g., 2′-O-ME ribonucleotides.

It will be understood by one of ordinary skill in the relevant arts thatother suitable modifications and adaptations to the methods,compositions, reaction mixtures and kits described herein are readilyapparent from the description of the invention contained herein in viewof information known to the ordinarily skilled artisan, and may be madewithout departing from the scope of the invention or any embodimentthereof. Having now described the present invention in detail, the samewill be more clearly understood by reference to the following examples,which are included herewith for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES

Examples are provided below illustrating certain aspects and embodimentsof the invention. The examples below are believed to accurately reflectthe details of experiments actually performed, however, it is possiblethat some minor discrepancies may exist between the work actuallyperformed and the experimental details set forth below which do notaffect the conclusions of these experiments or the ability of skilledartisans to practice them. Skilled artisans will appreciate that theseexamples are not intended to limit the invention to the specificembodiments described therein. Additionally, those skilled in the art,using the techniques, materials and methods described herein, couldeasily devise and optimize alternative amplification systems forcarrying out these and related methods while still being within thespirit and scope of the present invention.

Unless otherwise indicated, oligonucleotides and modifiedoligonucleotides in the following examples were synthesized usingstandard phosphoramidite chemistry, various methods of which are wellknown in the art. For example, using the Expedite.sup.™ 8909 DNASynthesizer (PerSeptive Biosystems, Framingham, Mass.). See e.g.,Carruthers et al. (1987) Meth. Enzymol. 154, 287. Unless otherwisestated herein, modified nucleotides were 2′-O-ME ribonucleotides, whichwere used in the synthesis as their phosphoramidite analogs. Reagentsand protocol conditions used in the performed experiments, as well as adiscussion of the results and conclusions of the experiments, are setforth below. The blocking moiety, if present, is at the 3′-end.

Example 1 Selective Hybridization and Capture of Target Nucleic Acids

In a first example, target capture was performed using a linear targetcapture oligomer and an inactivatable target capture oligomer. Theoligomers are presented in Table 1, below. The target nucleic acid was aP. acnes 16S ribosomal nucleic acid. The sequence of this target nucleicacid is found at GenBank Accession No.: AB042288.1 gi:7707831, firstseen at NCBI on May 5, 2000, with non-sequence updates on Jul. 22, 2002,Jan. 14, 2004 and Aug. 9, 2006, and is also SEQ ID NO:38 in the SequenceListing. To determine the efficiency of these target capture oligomers,captured nucleic acids were assayed in a subsequent real-time reverseTMA reaction (See, e.g., U.S. Pat. Nos. 5,480,784 and 5,399,491.).Briefly, a series of preannealing mixtures was made at 150.micro.Lvolume and comprising 0.5M LiCl lysis buffer, one of the linear targetcapture oligomer or the inactivatable target capture oligomer SEQ IDNOS:32 or 26, respectively, (6 pM/r×n each); a nonT7 primeramplification oligomer SEQ ID NO:39 (2 pm/r×n); and a terminatingoligomer SEQ ID NO:37 (2 pM/r×n). Here, the terminating oligomercomprised 2′-O-Me residues and both of the linear target captureoligomer and the inactivatable target capture oligomer comprised targethybridization regions comprising 2′-O-Me residues. Following a shortinitial incubation, 1×10.sup.4 copies of SEQ ID NO:38 was added to somewells as a positive control, and the remaining reaction wells contained0 copies per reaction of SEQ ID NO:38 for negative controls and forchallenge reactions. The reactions were then incubated for about 45minutes at about 60.deg.C followed by a 45 minute cool down at roomtemperature. Each reaction was then transferred to a microplatecontaining 50.micro.L of target capture reagent comprising a magneticbead comprising an immobilized probe. Target capture reagent for thereaction designated as challenge reactions were spiked with either1×10.sup.6 or 1×10.sup.7 copies of SEQ ID NO:38. Six replicates were runfor each condition.

A target capture protocol was performed essentially as follows. Targetcapture reactions were slowly mixed for about 5 minutes and then thebeads were collected and transferred to a first wash reaction. Capturedbeads were eluted into the first wash reaction, slowly mixed for about 5minutes, and the captured and eluted into a second wash reaction. Secondwash reactions were mixed; the beads were captured and then eluted intoa real-time TMA reaction mix. This amplification mix comprised a T7promoter provider SEQ ID NOS:33, detection probe SEQ ID NO:35 and anon-T7 amplification oligomer, SEQ ID NO:34, that hybridizes with thetag sequence of SEQ ID NO:39. In this example the detection probecomprised 2′-O-Me residues, a 5′ fluorescein, a 3′ Dabcyl and a spacerbetween residues 18 and 19. Components and conditions for reactionmixtures and incubation conditions are known in the art. Theamplification reaction was a real time RTMA reaction: pre-incubation at42.deg.C for 5 minutes on a thermomixer (Eppendorf Cat. No. 5355 34511);add 10 enzyme reagent; shake for 1 minute; and incubate at 42.deg.C.Results are below.

TABLE 1 SEQ ID NO: Sequence 5′→3′ 26GCUGAUAAGCCGCGAGUUAUCAGCtttaaaaaaaaaaa aaaaaaaaaaaaaaaaaaa 32GCUGAUAAGCCGCGAGUtttaaaaaaaaaaaaaaaaaa aaaaaaaaaaaa 33aatttaatacgactcactatagggagaCGAACGCTGGC GGCGTGCTTAACACATGC 34GTGACCCAATGATCTAACA 35 cCAGGGCCUUUCCGUUCGccugg 37 AGCGUUCGUCCUGAGCC 39gtgacccaatgatctaacagagcaccccacaaaagcag

TABLE 2 TTime RFU (min) Avg Condition Avg ∀SD SEQ ID NO: 26 >60.0  0.031No target/no challenge ∀0.003 SEQ ID NO: 26 19.5  0.293 1E4 target/nochallenge ∀0.009 SEQ ID NO: 26 24.89 0.281 No target/1E6 challenge∀0.010 SEQ ID NO: 26 21.27 0.286 No target/1E7 challenge ∀0.010 SEQ IDNO: 32 29.67 0.029 No target/no challenge ∀0.002 SEQ ID NO: 32 19.090.289 1E4 target/no challenge ∀0.009 SEQ ID NO: 32 21.4  0.297 Notarget/1E6 challenge ∀0.008 SEQ ID NO: 32 17.76 0.284 No target/1E7challenge ∀0.021

Results using the linear target capture oligo SEQ ID NO:32, werepositive and robust for all positive control reactions at 1×10.sup.4copies of SEQ ID NO:38 per reaction with an Average TTime=19.1 minutes.Two of the six negative samples produced weakly positive results. Allsix of the negative samples challenged with 1×10.sup.6 copies perreaction of P. acnes RNA during the target capture incubation stepproduced positive and robust results, as did all six of the negativesamples challenged with 1×10.sup.7 copies per reaction of P. acnes RNAduring the target capture incubation step. Thus, the linear targetcapture oligo was very susceptible to contaminating nucleic acidspresent in the target capture step. Results for the inactivatable targetcapture oligomer gave positive and robust results for 1×10.sup.4 copiesof P. acnes (Average TTime=19.5 minutes). No positive amp activity wasdetected for the six negative samples. Here too were all six of thenegative samples challenged with 1×10.sup.6 copies per reaction and allsix of the negative samples challenged with 1×10.sup.7 copies perreaction of P. acnes RNA positive. However, the results from thechallenge reactions were not as robust in the presence of theinactivatable target capture oligomer as they were in the presence ofthe linear target capture oligomer. Thus, the inactivatable targetcapture oligomer reaction wells discriminated surprisingly well againstcontaminating nucleic acids in the target capture reagent better thandid the linear amplification oligomer; even when the contaminatingnucleic acids are identical to the target nucleic acid.

Example 2 Selective Hybridization and Capture of Target Nucleic Acidsand Amplification with Heterologous Amplification Oligomers

A second set of experiments was conducted to compare the use of aninactivatable target capture oligomer to the use of a linear targetcapture oligomer; each along with a downstream amplification assay thatuses a heterologous amplification oligomer. Amplification assays usingheterologous amplification oligomers are described herein and are alsoknown in the art (e.g., Becker et al., U.S. Pub. No. 2007-0281317 A1).Preannealing reactions were set up generally as described above forexample 1. Oligomers used in the preannealing mixture were one of thelinear target capture oligomer or the inactivatable target captureoligomer SEQ ID NOS:32 or 26, respectively, (6 pM/r×n each); aheterologous amplification oligomer SEQ ID NO:36 (2 pm/r×n); and aterminating oligomer SEQ ID NO:37 (2 pM/r×n). Following a short initialincubation, 1×10.sup.4 copies of SEQ ID NO:38 was added to some wells asa positive control, and the remaining reaction wells contained 0 copiesper reaction of SEQ ID NO:38 for negative controls and for challengereactions. The reactions were then incubated for about 45 minutes atabout 60.deg.C followed by a 45 minute cool down at room temperature.Each reaction was then transferred to a microplate containing 50.micro.Lof target capture reagent comprising a magnetic bead comprising animmobilized probe. Target capture reagent for the reaction designated aschallenge reactions were spiked with either 1×10.sup.6 or 1×10.sup.7copies of SEQ ID NO:38. Six replicates were run for each condition.

A target capture protocol was performed essentially as follows. Targetcapture reactions were slowly mixed for about 5 minutes and then thebeads were collected and transferred to a first wash reaction. Capturedbeads were eluted into the first wash reaction, slowly mixed for about 5minutes, and the captured and eluted into a second wash reaction. Secondwash reactions were mixed; the beads were captured and then eluted intoa real-time TMA reaction mix. This amplification mix comprised a T7promoter provider SEQ ID NOS:33, an amplification oligomer comprising atarget hybridizing region that stably hybridizes the tag sequence of SEQID NO:36, that amplification oligomer being SEQ ID NO:34 and detectionprobe SEQ ID NO:35. Components and conditions for reaction mixtures andincubation conditions are known in the art. The amplification reactionwas a real time RTMA reaction: pre-incubation at 42.deg.C for 5 minuteson a thermomixer (Eppendorf Cat. No. 5355 34511); add 10 enzyme reagent;shake for 1 minute; and incubate at 42.deg.C. Results are below.

TABLE 3 SEQ ID NO: Sequence 5′→3′ 26GCUGAUAAGCCGCGAGUUAUCAGCtttaaaaaaaaaaa aaaaaaaaaaaaaaaaaaa 32GCUGAUAAGCCGCGAGUtttaaaaaaaaaaaaaaaaaa aaaaaaaaaaaa 33aatttaatacgactcactatagggagaCGAACGCTGGC GGCGTGCTTAACACATGC 34GTGACCCAATGATCTAACA 35 cCAGGGCCUUUCCGUUCGccugg 36CTGCTTTTGTGGGGGTGACCCAATGATCTAACACTCGA GCACCCCACAAAAGCAG 37AGCGUUCGUCCUGAGCC

TABLE 4 TTime RFU (min) Avg Condition Avg ∀SD SEQ ID NO: 26 >60.0  0.031No target/no challenge ∀0.001 SEQ ID NO: 26 23.77 0.298 1E4 target/nochallenge ∀0.003 SEQ ID NO: 26 31.29 0.029 No target/1E6 challenge∀0.002 SEQ ID NO: 26 31.48 0.051 No target/1E7 challenge ∀0.017 SEQ IDNO: 32 >60.0  0.031 No target/no challenge ∀0.003 SEQ ID NO: 32 22.390.291 1E4 target/no challenge ∀0.009 SEQ ID NO: 32 31.01 0.196 Notarget/1E6 challenge ∀0.083 SEQ ID NO: 32 25.39 0.286 No target/1E7challenge ∀0.005

Results using the heterologous amplification oligomer SEQ ID NO:36 withthe linear target capture oligo SEQ ID NO:32 were positive and robustfor all positive control reactions at 1×10.sup.4 copies of SEQ ID NO:38per reaction with an Average TTime=22.4 minutes. No positive ampactivity was detected for the six negative samples. Five out of six ofthe negative samples challenged with 1×10.sup.6 copies per reaction ofP. acnes RNA during the target capture incubation step produced weaklypositive amplification results. All six of the negative sampleschallenged with 1×10.sup.7 copies per reaction of P. acnes RNA duringthe target capture incubation step produced positive amps.

Results using the heterologous amplification oligomer SEQ ID NO:36 alongwith the inactivatable target capture oligo SEQ ID NO:26 were positiveand robust for all positive control reactions having 1×10.sup.4 copiesper reaction of P. acnes RNA; Average TTime=23.8 minutes. No positiveamp activity was detected for the six negative samples. One out of sixof the negative samples challenged with 1×10.sup.6 copies per reactionof P. acnes RNA during the target capture incubation step produced aweakly positive amplification. All six of the negative sampleschallenged with 1×10.sup.7 copies per reaction of P. acnes RNA duringthe target capture incubation step produced no amplification activity.Thus, the inactivatable target capture oligomer when used in conjunctionwith a heterologous amplification oligomer produced robust results anddiscriminated surprisingly well against contaminating nucleic acids inthe target capture reagent better than did the linear amplificationoligomer; even when the contaminating nucleic acids are identical to thetarget nucleic acid.

Example 3 Inactivatable Target Capture Oligomers Comprising Tag-ClosingRegions of Varying Length

A series of amplification and detection reactions were set-up usingeither a linear target capture oligomer or an inactivatable targetcapture oligomer. The target organism was P. acnes and the targetnucleic acid sequence was the 16S rRNA gene of P. acnes, SEQ ID NO:38.In a first set of experiments, inactivatable target capture oligomerswere designed to comprise tag-closing regions that are complementary toa target hybridizing portion of the capture probe. The tag-closingregions were designed to vary from 6 contiguous nucleobases in length to14 contiguous nucleobases in length. The inactivatable target captureoligomers also comprised a target hybridization region and a dT3A30nucleic acid sequence binding pair member. In this first experiment,these various inactivatable target capture oligomers were comparedagainst each other and against a linear target capture oligomer fortheir hybridization sensitivity towards SEQ ID NO:38. The linear targetcapture oligomer and the inactivatable target capture oligomers used inthese experiments are illustrated in Table 5.

TABLE 5 SEQ ID NO: Sequence 5′→3′ 25 GCUGAUAAGCCGCGAGUAUCAGC[TAIL] 26GCUGAUAAGCCGCGAGUUAUCAGC[TAIL] 27 GCUGAUAAGCCGCGAGUUUAUCAGC[TAIL] 28GCUGAUAAGCCGCGAGUCUUAUCAGC[TAIL] 29 GCUGAUAAGCCGCGAGUGCUUAUCAGC[TAIL] 30GCUGAUAAGCCGCGAGUCGGCUUAUCAGC[TAIL] 31GCUGAUAAGCCGCGAGUCGCGGCUUAUCAGC[TAIL] 32 GCUGAUAAGCCGCGAGU[TAIL]

In Table 5, the notation “[tail]” refers to the binding pair member thatis a dT3A30 nucleic acid sequence. In the embodiment of the currentexample, the non-tail residues are all 2′-O-Me residues. Underlinedresidues are the nucleic acid residues comprising the tag-closingregion, also referred to as a tag region, and which are configured tonot form a stable hybridization complex with the target nucleic acidsequence, but which will stably hybridize with the a portion of thetarget hybridization region of the target capture oligomer, therebyforming a loop structure and inactivating the target capture oligomerfrom further target capture. The target hybridizing regions, tag-closingregions and tails (or target hybridizing region and tail for SEQ IDNO:32) are joined as contiguous nucleic acid sequences withoutnon-nucleotide linkers.

In addition to one of the target capture oligomers listed in Table 5,oligomers used in these reactions included a terminating oligomer SEQ IDNO:37, a tagged primer oligomer SEQ ID NO:36, a primer oligomer thathybridizes the tag region of SEQ ID NO:36, that primer oligomer beingSEQ ID NO:34, a promoter-based oligomer SEQ ID NO:33, and a detectionprobe oligomer SEQ ID NO:35. Preferably, SEQ ID NO:33 is blocked, thusit is a promoter provider. See, Table 6 illustrating these additionaloligomers

TABLE 6 SEQ ID NO: Sequence 5′→3′ 33aatttaatacgactcactatagggagaCGAACGCTGGC GGCGTGCTTAACACATGC 34GTGACCCAATGATCTAACA 35 cCAGGGCCUUUCCGUUCGccugg 36CTGCTTTTGTGGGGGTGACCCAATGATCTAACACTCGA GCACCCCACAAAAGCAG 37AGCGUUCGUCCUGAGCC

In Table 6, the lower case letters of SEQ ID NO:33 represent thepromoter region; the lower case letters of SEQ ID NO:35 represent thestem forming region and the underlined letters of SEQ ID NO:36 representthe tag region. Further, SEQ ID NO:35 comprises 2′-O-Me residues, a 5′fluorescein at residue number 1, a spacer between residues 18 and 19,and a 3′ dabcyl following residue number 23. SEQ ID NOS:33 and 37 are 3′blocked.

A series of preannealing mixtures were made at 150.micro.L volume andcomprising 0.5M LiCl lysis buffer, SEQ ID NOS:36 and 37 (2 pM/r×n each)and one of SEQ ID NOS:25-32 (6 pM/r×n each). Each of these reactionconditions was combined with one or more of either 1×10.sup.3,1×10.sup.4 or 1×10.sup.5 copies per reaction of P. acnes 16S ribosomalnucleic acid (SEQ ID NO:38). Negative control reactions were also madeto include 0 copies per reaction of P. acnes target nucleic acid. Atarget capture protocol was performed essentially as follows. Targetcapture reactions were slowly mixed for about 5 minutes and then thebeads were collected and transferred to a first wash reaction. Capturedbeads were eluted into the first wash reaction, slowly mixed for about 5minutes, and the captured and eluted into a second wash reaction. Secondwash reactions were mixed; the beads were captured and then eluted intoa real-time rTMA reaction mix. This amplification mix comprisedamplification oligomers SEQ ID NOS:33 and 34 and detection probe SEQ IDNO:35. Components and conditions for reaction mixtures and incubationconditions are known in the art (See, e.g., U.S. Pat. Nos. 5,480,784 and5,399,491.) Results are presented in Table 7.

TABLE 7 TTime RFU (min) Avg Condition Avg ∀SD SEQ ID NO: 32 and 28.640.202 0 copies of SEQ ID NO: 38 ∀0.18815698 SEQ ID NO: 32 and 23.310.471083333 1e4 copies of SEQ ID NO: 38 ∀0.032424461 SEQ ID NO: 32 and22.55 0.50455 1e5 copies of SEQ ID NO: 38 ∀0.027238998 SEQ ID NO: 29 and28.03 0.023309091 0 copies of SEQ ID NO: 38 ∀0.003551184 SEQ ID NO: 29and 27.52 0.024325 1e4 copies of SEQ ID NO: 38 ∀0.001703673 SEQ ID NO:29 and 28.17 0.352683333 1e5 copies of SEQ ID NO: 38 ∀0.078898046 SEQ IDNO: 30 and 27.19 0.025636364 0 copies of SEQ ID NO: 38 ∀0.002111527 SEQID NO: 30 and 29.46 0.208833333 1e4 copies of SEQ ID NO: 38 ∀0.164515102SEQ ID NO: 30 and 28.90 0.40535 1e5 copies of SEQ ID NO: 38 ∀0.069912882SEQ ID NO: 31 and >60.0  0.025645455 0 copies of SEQ ID NO: 38∀0.002715645 SEQ ID NO: 31 and 27.96 0.206666667 1e4 copies of SEQ IDNO: 38 ∀0.195675994 SEQ ID NO: 31 and 29.88 0.142883333 1e5 copies ofSEQ ID NO: 38 ∀0.157650035 SEQ ID NO: 25 and >60.0  0.028166667 0 copiesof SEQ ID NO: 38 ∀0.001661507 SEQ ID NO: 25 and 26.78 0.21482 1e4 copiesof SEQ ID NO: 38 ∀0.003179029 SEQ ID NO: 25 and 24.10 0.2277 1e5 copiesof SEQ ID NO: 38 ∀0.011768602 SEQ ID NO: 26 and >60.0  0.031475 0 copiesof SEQ ID NO: 38 ∀0.002243105 SEQ ID NO: 26 and 27.38 0.4149 1e4 copiesof SEQ ID NO: 38 ∀0.026926769 SEQ ID NO: 26 and 24.04 0.408 1e5 copiesof SEQ ID NO: 38 ∀0.024519973 SEQ ID NO: 27 and 22.96 0.044113043 0copies of SEQ ID NO: 38 ∀0.003205153 SEQ ID NO: 27 and 26.59 0.6035833331e4 copies of SEQ ID NO: 38 ∀0.024796805 SEQ ID NO: 27 and 23.810.612841667 1e5 copies of SEQ ID NO: 38 ∀0.029039173 SEQ ID NO: 28 and12.76 0.046845833 0 copies of SEQ ID NO: 38 ∀0.003918959 SEQ ID NO: 28and 24.54 0.578190909 1e4 copies of SEQ ID NO: 38 ∀0.020016966 SEQ IDNO: 28 and 22.49 0.595809091 1e5 copies of SEQ ID NO: 38 ∀0.036150338

The results presented in Table 7 illustrate that the inactivatabletarget capture oligomers all had reduced background fluorescence whencompared to that of the linear capture probe (compare the reaction wellsfor 0 copies of target nucleic acid). The samples with linear captureprobe provided an average of 0.2 RFU. Samples with inactivatable targetcapture oligomers provided on average about 0.02 RFU for inactivatabletarget capture oligomers having longer tag-closing regions (e.g., 10 to14 nucleobases in length) to about 0.04 RFU for those with shortertag-closing regions (e.g., 7-9 nucleobases in length). The longertag-closing regions also resulted in longer average TTimes than did theshorter tag-closing regions; compare, Table 7 results for inactivatabletarget capture oligomers comprising tag-closing regions of 10 to 14nucleobases in length with inactivatable target capture oligomerscomprising tag-closing regions of 6 to 9 nucleobases in length.Surprisingly, these results show no linear relationship between thelength of the tag-closing region and the average TTime. Similarly, forthe average RFUs, the inactivatable target capture oligomers comprisingshorter tag-closing regions (6 to 9 nucleobases in length) providedhigher average RFUs than did the inactivatable target capture oligomerscomprising longer tag-closing regions (10 to 14 nucleobases in length).(See, Table 7). Again, there is surprisingly no linear relationshipbetween the length of the tag region and the resultant data.

In a second set of reactions, the inactivatable target capture oligomersof SEQ ID NO:26 and the linear capture probe of SEQ ID NO:32 are used ina series of amplification reactions in the presence or absence of achallenge organism. The reactions were set up substantially as describedabove and comprised a terminating oligomer, a heterologous amplificationoligomer, a T7 promoter provider oligomer, a primer targeting the tagsequence of the heterologous amplification oligomer and a detectionprobe oligomer of Table 6. The P. acnes target nucleic acid was SEQ IDNO:38. The challenge nucleic acid was also P. acnes SEQ ID NO:38.Preannealing reactions contained: either 1×10.sup.4 copies of targetnucleic acid as a positive control or 0 copies of target nucleic acid;either 6 pM/reaction of linear target capture oligomer or 6 pM/reactionof inactivatable target capture oligomers; 2 pM/reaction of blockeroligomer; and 2 pM/reaction of heterologous amplification oligomer. Aseries of target capture reagent mixtures were made to include1×10.sup.5; 1×10.sup.6 or 1×10.sup.7 copies of P. acnes challengenucleic acid. Following the preannealing step, the preannealingreactions with 0 copies of target nucleic acid were combined with eachone of the target capture mixtures. The positive control preannealingreaction mixture was combined with a target capture mixture containing 0copies of challenge nucleic acid. A target capture incubation step wasthen performed on each reaction condition, followed by a wash step andthen an amplification step. Amplification was TMA, as generallydescribed herein. Results are shown in Table 8.

TABLE 8 TTime RFU (min) Avg Condition Avg ∀SD SEQ ID NO: 32 >60.0 0.016466667 0 copies of challenge ∀0.003868161 SEQ ID NO: 32 21.830.435566667 E4 copies of target ∀0.051765883 SEQ ID NO: 32 >60.0 0.022641667 E5 copies of challenge ∀0.005540669 SEQ ID NO: 32 36.610.024166667 E6 copies of challenge ∀0.005970343 SEQ ID NO: 32 28.220.281641667 E7 copies of challenge ∀0.138143604 SEQ ID NO: 26 >60.0 0.0159 0 copies of challenge ∀0.001649242 SEQ ID NO: 26 21.36 0.427 E4copies of target ∀0.034371645 SEQ ID NO: 26 >60.0  0.021975 E5 copies ofchallenge ∀0.004562719 SEQ ID NO: 26 21.99 0.020954545 E6 copies ofchallenge ∀0.005131348 SEQ ID NO: 26 35.90 0.021488889 E7 copies ofchallenge ∀0.003267432

These results indicate that the inactivatable target capture oligomershows better resistance to challenge organism compared to the linearcapture probe. For both probes, the negative control reaction conditions(0 copies of challenge and 0 copies of target) had negative RFU values.Positive control reaction conditions (0 copies challenge and 1×10.sup.4target) had positive RFU values for both. The linear probe, though,showed positive RFU values for in the presence of challenge organism.With the exception of a single positive reaction well seen in the1×10.sup.6 conditions, the inactivatable target capture oligomers didnot show positive RFU values.

Example 4 Selective Amplification of HCV Using Tagged Oligonucleotidesin a Real-Time TMA Reaction

The following series of experiments were conducted to evaluate whetherthe use of a tagged oligonucleotide to modify a target nucleic acidsequence in a nucleic acid sample of interest prior to atranscription-mediated amplification reaction would permit the selectiveamplification of target nucleic acid sequence contributed by the nucleicacid sample of interest, while not amplifying target nucleic acidsequence contributed by sources other than the nucleic acid sample ofinterest.

TABLE 9 Oligonucleotides SEQ ID NO: Sequence 5′→3′ Comment 1GTTTGTATGTCTGTTGCTATTATGTCTACAGGCATTGAGCGG Tagged GTTGATCCAAGAAAGGACOligomer. 12 pM/rxn 2 GTTTGTATGTCTGTTGCTATTAT Priming Oligomer.12 pM/rxn 3 ATTTAATACGACTCACTATAGGGAGACCACAACGGTTTCTA PromoterGCCATGGCGTTAGTATGAG-blocking moiety Oligomer 12 pM/rxn 4AmUmGmGmCmUmAmGmAmCmGmCmUmUmUmCmUmGmCmGmUm TerminatingGmAmAmGmAm-blocking moiety Oligomer m = 2′-Ome 0.8 pM/rxn 5TGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATA- Extending blocking moietyOligomer. 12 pM/rxn 6 GmGmGmCmAmCmUmCmGmCmAmAmGmCmAmmCmCmCmUmTTLinear capture TAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Probe 3 pM/rxn. m = 2′Ome7 CmAmUmGmGmUmGmCmAmCmGmGmUmCmUmAmCmGmTTTAA Linear captureAAAAAAAAAAAAAAAAAAAAAAAAAAAA Probe 3 pM/rxn. m = 2′Ome 8¹CmGmUmUmCmCmGmCmAmGmAmCmCmAmCmUmAmUm²GmAm Detection AmCmGm³ Probe.4 pM/rxn m = 2′-Ome

Blocking Moiety: A 3′-to-3′ linkage prepared using3′-dimethyltrityl-N-benzoyl-2′-deoxycytidine, 5′-succinoyl-long chainalkylamino-CPG (Glen Research Corporation, Sterling, Va.; Cat. No.20-0102-01). 1=6-Carboxyfluorescein (FAM or fluorescein) (BioGenex, SanRamon, Calif.; Cat. No. BGX-3008-01); 2=9-O-Dimethoxytrityl-triethyleneglycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (C9 linker)(Glen Research Corporation, Sterling, Va.; Cat. No. 10-1909-90); and3=4-(4′-dimethylaminophenylazo) benzoic acid (DABCYL) (Prime Synthesis,Inc., Aston, Pa.).

Reagents and Other Protocol Information. Amplification Reagent. The“Amplification Reagent” or “AMP Reagent” comprised 11.6 mM TrizmaR basebuffer, 15 mM TrizmaR hydrochloride buffer, 25 mM MgCl.sub.2, 23.3 mMKCl.sub.2, 3.33% (v/v) glycerol, 0.05 mM zinc acetate, 0.76 mM dATP,0.76 mM dCTP, 0.76 mM dGTP, 0.76 mM dTTP, 0.02% (v/v) ProClin 300Preservative (Supelco, Bellefonte, Pa.; Cat. No. 48126), 6.0 mM ATP, 6.0mM CTP, 6.0 mM GTP, and 6.0 mM UTP, pH 7.81 to 8.0 at 22.deg.C. EnzymeReagent. The “Enzyme Reagent” comprised 70 mM N-acetyl-L-cysteine, 10%(v/v) TRITON.sup.R X-102 detergent, 16 mM HEPES, 3 mM EDTA, 0.05% (w/v)sodium azide, 20 mM Trizma.sup.R base buffer, 50 mM KCl.sub.2, 20% (v/v)glycerol, 165.6 mM trehalose, pH 7, and containing 224 RTU/.micro.LMoloney murine leukemia virus (“MMLV”) reverse transcriptase and 140U/.micro.L T7 RNA polymerase, where one unit (i.e., RTU or U) ofactivity is defined as the synthesis and release of 5.75 fmol cDNA in 15minutes at 37.deg.C for MMLV reverse transcriptase, and the productionof 5.0 fmol RNA transcript in 20 minutes at 37.deg.C for T7 RNApolymerase. Wash Solution. The “Wash Solution” comprised 10 mM HEPES,6.5 mM NaOH, 1 mM EDTA, 0.3% (v/v) ethyl alcohol, 0.02% (w/v) methylparaben, 0.01% (w/v) propyl paraben, 150 mM NaCl, and 0.1% (w/v) sodiumdodecyl sulfate, pH 7.5. Transport Medium. The “Transport Medium”comprised 150 mM HEPES, 8% (w/v) lithium lauryl sulfate, and 100 mMammonium sulfate, pH 7.5. Target Capture Reagent. The “Target CaptureReagent” or “TCR” comprised the components listed below. Additionalinformation about the formulation of this mixture is described belowunder Target Capture Reagent Procedure (IIIA). The concentrations listedrepresent the final concentrations of the components after having beencombined with the magnetic particle solution. The magnetic particleswere Sera-Mag.sup.™ MG-CM Carboxylate Modified (Seradyn, Inc.,Indianapolis, Ind.; Cat. No. 24152105-050250), 1 micron,super-paramagnetic particles covalently bound 5′-amino modifiedoligo(dT).sub.14. The HEPES, lithium hydroxide, lithium chloride, EDTA,lithium lauryl sulfate and ammonium sulfate components were introducedwith the TCR diluent and Transport Medium. First Capture Probe; 15.0 nM;Second Capture Probe; 15.0 nM; Tagged Priming Oligonucleotide; 60.0 nM;Terminating Oligonucleotide; 4.0 nM; HEPES, Free Acid, Dihydrate; 118.7mM; Lithium Hydroxide, Monohydrate; 98.9 mM; Lithium Chloride, HighPurity; 470.6 mM; EDTA, Free Acid; 25.0 mM; Lithium Lauryl Sulfate;110.2 mM; Ammonium Sulfate; 37.5 mM; and Seradyn Poly dT14 MagneticParticles; 0.075.micro.g/uL. Transcript Buffer. The “Transcript Buffer”comprised 0.2% lithium lauryl sulfate. Transcript Used. HCV Transcript.Product Numbers of Certain Materials or Equipment Used. KingFisher.sup.™Plate (Thermo Labsystems, Franklin, Mass.; Cat. No. 97002540); MJResearch microtiter plate (Bio-Rad Laboratories, Inc., Hercules, Calif.;Cat. No. HSP-9665); Solo HT Incubator (Thermo Labsystems, Franklin,Mass.; Cat. No. 5161580); KingFisher.sup.™ Comb (Thermo Labsystems,Franklin, Mass.; Cat. No. 97002510); Eppendorf.sup.® Thermomixer R(Eppendorf North America; Westbury, N.Y.; Cat. No. 022670107 or022670158)′ and DNA Engine Opticon.sup.® 2 Real-Time PCR DetectionSystem (Bio-Rad Laboratories, Inc., Hercules, Calif.; Cat. No.CFB-3220).

Additional Protocol Information. For the described experiments,3.3.micro.L of target-containing transcript buffer was added to each 2.0ml microtube in step B6 below. The tagged priming oligonucleotide andthe terminating oligonucleotide were in water before being added to the2.0 mL microtubes. Samples were vortexed for about 5 seconds. Incubatingfor 10 minutes at 60.deg.C was found to be generally sufficient tocapture the transcript. The plates were kept at room temperature for 5minutes following the 10-minute incubation to allow the plates to coolbefore the target capture steps. This is also where the plates weretransferred from the Solo HT Incubator to the KingFisher System. Thespeed of the thermomixer was 1400 rpm.

Target Capture Protocol: Target Capture Reagent (TCR) Procedure.Magnetic beads were slowly mixed at room temperature (RT) for 45 minutesand 150.micro.L magnetic beads were added to 5 mL TCR diluent(15.micro.g beads/r×n when 50.micro.L used per sample). The solution wasslowly mixed at room temperature for 35-minutes, at which time captureprobe was added to 5 mL of the TCR diluent (to a final concentration of0.12 pmol/.micro.L (6-pmol/50.micro.L r×n). Sample Preparation: AMPReagent was prepared containing the promoter oligonucleotide, extenderoligonucleotide and priming oligonucleotide (volume=1,600.micro.L). Thesolution was vortexed and placed at 2-8.deg.C until needed. Detectionprobe was prepared in Enzyme Reagent and placed at 2-8.deg.C untilneeded. Target dilutions were prepared in 0.2% LLS. 50.micro.L TCR wastransferred into 200.micro.L microplate wells. Each target copy level,tagged priming oligonucleotide and terminating oligonucleotide wereadded to 1.2 mL 50% Transport Medium, 50% H2O in 2.0 mL microtubes.Target samples were vortexed and 150.micro.L transferred into200.micro.L microplate (Plate 1) well containing 50.micro.L TCR (eachwell contained zero or 1 million copies HCV transcript plus appropriateamounts of tagged priming and terminating oligonucleotides).

Target Capture Protocol. The 200.micro.L microplate (Plate 1) wasincubated at 60.deg.C for 10 minutes using Labsystems Solo HT Incubator(Plate 1), and the microplate was then placed at RT for 5 minutes (Plate1). 200.micro.L microplates (Plates 2 & 3) were prepared with200.micro.L Wash Reagent. Amplification plate (Plate 4-MJ research 96well microtiter plate) was prepared with 30 .micro.L AMP Reagent perwell. The 96 well comb was placed into Plate 1. All four plates wereloaded into the KingFisher 96 unit and the target capture protocol wasinitiated, as follows. Plate 1 was mixed for 5 minutes at very slowspeed and beads were collected for 12 counts and then released intoPlate 2 for 10 seconds using slow speed. Plate 1 was then mixed for 1second using very slow speed, beads collected for 12 counts, and thebeads were released into Plate 2 for 10 seconds using slow speed. Plate2 was mixed for 30 seconds at medium speed and beads were collected for12 counts and then released into Plate 3 for 10 seconds using very slowspeed. Plate 2 was then mixed for 1 second at very slow speed and beadswere collected for 12 counts and released into Plate 3 for 10 secondsusing very slow speed. Plate 3 was mixed for 30 seconds at medium speed,beads were collected for 12 counts, and the beads were released intoPlate 4 for 10 seconds using medium speed. Plate 3 was then mixed for 1second at very slow speed, beads collected for 12 counts and releasedinto plate 4 for 10 seconds using medium speed. The 96 well microtiterplate (Plate 4) was removed and transferred to the bench, covered with asealing card, and placed in the DNA Engine Opticon.sup.® 2 Real-Time PCRDetection System (Bio-Rad Laboratories; Hercules, Calif.) (“real-timeinstrument”). Real Time TMA was performed as follows. The plate wasincubated for 5 minutes at 42.deg.C and then removed and placed on a42.deg.C thermomixer. Each reaction well received a 10.micro.L aliquotof the Enzyme Reagent. The microtiter plate was covered with an adhesivetape seal, shaken gently for 30 seconds on the thermomixer, and thenplaced into the real-time instrument at 42.deg.C, where real-time assaymonitoring was commenced. TTime values, which served as indicators ofthe amount of amplicon synthesized, were determined from the monitoredfluorescence signals. See Light et al., U.S. Pub. No. US 2006-0276972,paragraphs 506-549.

Results and Conclusion: Experiments were performed according to theprocedures described above for detecting an HCV transcript (8replicates). The TCR in each test contained the same tagged primingoligonucleotide. A target capture step was performed for binding HCVtranscript and removing unhybridized tagged priming oligonucleotide andterminating oligonucleotide. After the target capture step, an AMPReagent was contacted with the beads of the TCR, with the AMP Reagentcontaining a priming oligonucleotide specific for the complement of thetag sequence. No tagged priming oligonucleotide was included in thisstep. Eight replicates were run for each condition. The detection probewas added via the Enzyme Reagent at 4 pmol per reaction. The HCV AMPReagent contained 12 pmol promoter oligonucleotide, 12 pmol extenderoligonucleotide and 12 pmol priming oligonucleotide per reaction.

The first set of experiments compared the results of reactions in whichno copies of the HCV transcript were spiked into the TCR or AMP Reagentwith the results of reactions in which 1×10.sup.6 copies of the HCVtranscript were spiked into the TCR. FIG. 17 shows the raw curves forHCV amplifications in which no target was spiked into the AMP Reagent.There was no detectable amplification when the HCV transcript was notspiked into the TCR or AMP Reagent, while the average TTime forreactions containing 1×10.sup.6 copies of the HCV transcript in the TCRwas 6.3 minutes. The “TTime” values relate to time of emergence (time atwhich signal rises above background), and a summary of these values forthe experiments performed is set forth in Table 1 below.

A second set of experiments compared the results of reactions in which1×10.sup.6 copies of the HCV transcript were spiked into the AMP Reagentonly with reactions in which 1×10.sup.6 copies of the HCV transcriptwere spiked into the TCR only. FIG. 18 shows the raw curves for HCVamplifications in which target was spiked into the AMP Reagent. Therewas no detectable amplification when the HCV transcript was spiked intothe AMP Reagent, while the average TTime for reactions containing 1×106copies of the HCV transcript in the TCR was 6.3 minutes (Table 1). Thezero target in TC samples did not amplify, even with 1 million copiesHCV transcript spiked into the AMP Reagent.

A third set of experiments compared the results of reactions in which1×10.sup.6 copies of the HCV transcript and the tagged primingoligonucleotide were provided in the AMP Reagent (no copies of the HCVtranscript in TCR) with the results of reactions in which 1×10.sup.6copies of the HCV transcript were provided in the TCR and the taggedpriming oligonucleotide was provided in the AMP Reagent. FIG. 19 showsthat the Average TTime for 1 million copies HCV transcript present onlyin the target capture step with tagged priming and terminatingoligonucleotides spiked into the AMP Reagent was 7.2 minutes. The zerosamples with target, terminating oligonucleotide and tagged primingoligonucleotide spiked into the AMP Reagent also produced robustamplification with an Average TTime=8.6 minutes (Table 9).

TABLE 9 TTime Summary (AvgTTimes & SDTTimes) Target Target Avg. SDTSample ID Name Amt Total RN1 TN1 TTime Time 1 million target in TC-x6.0HCV 1E6 8 7 8 6.3 0.11 1 million target in TC, tagged HCV 1E6 8 8 8 7.20.20 non-T7 primer & terminating oligonucleotide in amp-x6.0 1 milliontarget in TC-x6.0 HCV 1E6 8 8 7 6.3 0.05 Zero target in TC, 1 millionHCV 0.00 8 8 0 N/A N/A targ in amp-x0.0 Zero target in TC, 1 million HCV0.00 8 8 8 8.6 0.21 target, tagged non-T7 primer & terminatingoligonucleotide in amp-x0.0 Zero target in TC-x0.0 HCV 0.00 8 8 0 N/AN/A

The results of these experiments demonstrate that only when the taggedpriming oligonucleotide was present in the AMP Reagent along with thepriming oligonucleotide did zero TCR samples amplify when 1 millioncopies of HCV transcript were spiked into the AMP Reagent. Thus, HCVtranscript entering the system through the AMP Reagent is not amplifiedunless the tagged priming oligonucleotide is also provided with the AMPReagent.

The preceding Example demonstrated how a tagged priming oligonucleotidethat hybridized to an HCV template could be used for selectivelydetecting HCV nucleic acids in a sample of interest without interferencefrom contaminating nucleic acid introduced subsequent to a targetcapture step. The following Example illustrates how a similar approachwas used for detecting bacterial nucleic acids in a sample of interestdespite the presence of contaminating templates in reagents used forperforming the amplification reaction. Advantageously, non-complexedtagged priming oligonucleotide was substantially absent from thereaction mixture at the time a complex comprising the tagged primingoligonucleotide and the template contacted the DNA polymerase used inthe amplification reaction.

Example 5 below describes two procedures for amplifying E. coli rRNAnucleic acids, where the procedures differed by the use of both a taggedpriming oligonucleotide and target capture. The first procedure employedan E. coli specific non-tagged priming oligonucleotide in combinationwith a terminating oligonucleotide, a promoter oligonucleotide and adetection probe. The second procedure employed a tagged primingoligonucleotide having a target-complementary sequence identical to thatcontained in the E. coli specific non-tagged priming oligonucleotide ofthe first procedure, a tag-specific priming oligonucleotide, as well asa terminating oligonucleotide, a promoter oligonucleotide and adetection probe. The tag-specific priming oligonucleotide, which had anucleotide sequence corresponding to a segment of HIV-1, hybridized tothe complement of the tag sequence contained in the tagged primingoligonucleotide, but did not hybridize to the E. coli rRNA templatenucleic acid or the complement thereof. In the case of the secondprocedure, the terminating oligonucleotide, the promoter oligonucleotideand the detection probe were identical to those used in the firstprocedure. As demonstrated below, amplification reactions that omittedthe tagged priming oligonucleotide failed to discriminate betweensamples containing 0 and 1×10.sup.6 copies of a synthetic E. coli rRNAtarget. Conversely, the approach that included use of a tagged primingoligonucleotide and target capture clearly distinguished samplescontaining 0 and 1×10.sup.3 copies of the synthetic E. coli rRNA target.

Example 5 Use of a Tagged Priming Oligonucleotide Allows DiscriminationBetween Sample-Derived Templates and Exogenous Templates AmplificationUsing a Non-Tagged Priming Oligonucleotide without Target Capture

In a first procedure, amplification reactions employing a synthetic E.coli rRNA template were performed using a non-tagged primingoligonucleotide that hybridized to the template, a promoteroligonucleotide, a terminating oligonucleotide and a molecular torchdetection probe. Reactions were primed using the synthetic templateadded directly into the reaction mixtures (i.e., without undergoingtarget capture purification) at 0 or 1×10.sup.6 copies/reaction. Amolecular torch detection probe was used to monitor amplicon productionas a function of time. In the nucleotide sequences presented below,2′-O-methyl ribose (2′-O-Me) modifications of the polynucleotidebackbone are indicated by lower case “m.” Blocking moieties at the 3′termini of the promoter oligonucleotide and terminating oligonucleotidecomprised a 3′-to-3′ linkage that was prepared using3′-dimethyltrityl-N-benzoyl-2′-deoxycytidine, 5′-succinoyl-long chainalkylamino-CPG (Glen Research Corporation, Sterling, Va.; Cat. No.20-0102-01). Oligonucleotides, reagents and essential methods used inthe procedure were as follows.

TABLE 10 Oligonucleotides SEQ ID NO: Sequence 5′→3′ Comments  9CmUmGmCmTGGCACGGAGTTAGCCGGTGCTTC Priming Oligomer. m = 2′Ome 10ATTTAATACGACTCACTATAGGGAGAGAAGGCCTTCGGGTTG PromoterTAAAG-blocking moiety Provider 11 GmCmCmUmUmCmUmUmCmAmUmAmCmAmCmGmCmGm-Terminating blocking moiety Oligomer m = 2′Ome 12¹CmUmGmCmGmGmGmUmAmAmCmGmUmCmAmAmUmGmAmGmCm Detection AmAmAm²CGCAG³Probe 1 fluorescein 2 C9 linker 3 DABCYL

Reagents and Other Protocol Information: Amplification and enzymereagents were essentially as described under Example 4. Procedures usingthe non-tagged priming oligonucleotide that hybridized to the E. colitemplate did not employ target capture oligonucleotides or reagents, didnot employ transport medium or wash solution, and did not employ anextender oligonucleotide.

Real-Time Amplification Protocol. Sample solutions were prepared usingprimerless amplification reagent, non-tagged priming oligonucleotide,promoter oligonucleotide, terminating oligonucleotide, detection probeand synthetic template nucleic acid. Each well of a 96-well microtiterplate received a 30.micro.L aliquot of the prepared sample solution. Themicrotiter plate was covered with an adhesive tape seal, incubated firstfor 10 minutes at 60.deg.C in the DNA ENGINE OPTICON.sup.® 2 (Bio-RadLaboratories; Hercules, Calif.) temperature-controlled real-timeinstrument, and then temperature-adjusted to 42.deg.C for 5 minutes.Thereafter, the plate was removed from the real-time instrument andplaced onto a 42.deg.C thermomixer. Each reaction well received a10.micro.L aliquot of the enzyme reagent. The microtiter plate wascovered with an adhesive tape seal, shaken gently for 30 seconds on thethermomixer, and then placed into the real-time instrument at 42.deg.Cwhere real-time assay monitoring was commenced. TTime values, whichserved as indicators of the amount of amplicon synthesized, weredetermined from the monitored fluorescence signals.

Results and Conclusion

As indicated in FIG. 20, substantially identical results were observedin reactions that included either 0 or 1×10.sup.6 copies of the templatenucleic acid, and so the assay showed no discrimination between thesetwo conditions. More specifically, fluorescent signals indicatingformation of E. coli nucleic acid amplification products emerged frombackground levels at substantially similar times (i.e., TTime=31.74minutes at the 0 copy level, and 31.19 minutes at the 10.sup.6 copylevel) in both reactions. Thus, a real-time amplification profilecharacteristic of high levels of the nucleic acid template was obtainedeven in the absence of added E. coli rRNA template. This was consistentwith the presence of contaminating bacterial nucleic acid templates inone or more of the reagents used for carrying out the amplificationreactions following the target capture procedure.

Amplification Using a Tagged Priming Oligonucleotide and Target Capture

In a second procedure, a tagged priming oligonucleotide and targetcapture step were employed for performing amplification reactions usingtest samples containing either 0, 103 or 105 copies of the synthetic E.coli transcript. Oligonucleotides used in the procedure are indicatedbelow. The molecular torch detection probe was added as a component ofthe enzyme reagent. Following target capture, tagged primingoligonucleotide that was not hybridized to template nucleic acid wasremoved from the system by standard target capture and wash steps. Thecomplex that included the rRNA template and the tagged primingoligonucleotide remained captured on super-paramagnetic particles.Amplification reactions were carried out using reagents essentially asdescribed above, except for substitution of a non-specific targetcapture oligomer for the sequence-specific capture probes employed inExample 4. Amplification reactions were carried out in replicates of sixand monitored using a molecular torch detection probe essentially asdescribed in Example 4, except that an extender oligonucleotide wasomitted. As above, 2′-O-methyl ribose (2′-O-Me) modifications of thepolynucleotide backbone in the sequences presented below are indicatedby lower case “m.” Blocking moieties at the 3′ termini of the promoteroligonucleotide and terminating oligonucleotide comprised a 3′-to-3′linkage that was prepared using3′-dimethyltrityl-N-benzoyl-2′-deoxycytidine, 5′-succinoyl-long chainalkylamino-CPG (Glen Research Corporation, Sterling, Va.; Cat. No.20-0102-01). Oligonucleotides, reagents and essential methods used inthe procedure were as follows.

TABLE 10 Oligonucleotides SEQ ID NO: Sequence 5′→3′ Comments 14GTTTGTATGTCTGTTGCTATTATGTCTACCTGCTGGCACGGAG Tagged TTAGCCGGTGCTTC Primer15 GTTTGTATGTCTGTTGCTATTAT Tag-specific Primer 10ATTTAATACGACTCACTATAGGGAGAGAAGGCCTTCGGGTTG PromoterTAAAG-blocking moiety Provider 11 GmCmCmUmUmCmUmUmCmAmUmAmCmAmCmGmCmGm-Terminating blocking moiety Oligomer m = 2′Ome 16KmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmTTTAAAA Non-specificAAAAAAAAAAAAAAAAAAAAAAAAAA Capture Probe. m = 2′ Ome K = g or t\u 12¹CmUmGmCmGmGmGmUmAmAmCmGmUmCmAmAmUmGmAmGmCmAm Detection AmAm²CGCAG³Probe 1 fluorescein 2 C9 linker 3 DABCYL 13 See Sequence ListingSynthetic E. coli rRNA template

Reagents and Other Protocol Information: Reagents and experimentalprotocols were essentially as described under Example 4, with thesubstitution of a non-specific target capture oligonucleotide for thefirst and second capture oligonucleotides, the substitution of theabove-presented E. coli-specific oligonucleotides for HCV-specificoligonucleotides, and the omission of an extender oligonucleotide.Non-specific Target Capture Protocol: Target Capture Reagent (TCR)Preparation. A stock suspension of magnetic beads was mixed at roomtemperature for 30 minutes. An aliquot of about 150.micro.L of themagnetic bead suspension was added to 5 mL of TCR diluent (15.micro.gbeads/reaction when using 50.micro.L/sample), and then slowly mixed atroom temperature for 30 minutes. Next, the non-specific captureoligonucleotide was added to 5 mL of the TCR mixture to yield a finalconcentration of 0.12 pmol/.micro.L. The prepared TCR was mixed gentlyat room temperature until needed. Sample Preparation. Amplificationsolution was prepared using primerless amplification reagent, promoteroligonucleotide and tag-specific priming oligonucleotide. The preparedamplification solution was mixed by vortexing and then maintained at2-8.deg.C until needed. Enzyme reagent containing the molecular torchdetection probe was next prepared and maintained at 2-8.deg.C untilneeded. Dilutions of the template rRNA were prepared in 0.2% LLS(lithium lauryl sulfate). Aliquots (50.micro.L) of the magnetic beadtarget capture solution were transferred into the wells of a microtiterplate for a KINGFISHER 96 (Thermo Fisher Scientific, Inc.; Waltham,Mass.) magnetic particle processor. Samples of diluted template, taggedpriming oligonucleotide and terminating oligonucleotide were then addedto 1.5 mL of 50% transport medium diluted with water. Thetarget-containing sample mixture was vortexed, and 150.micro.L aliquotstransferred into the microtiter plate (Plate 1) wells containing 50.micro.L target capture solution (each well contained 0, 10.sup.3 or10.sup.5 copies of the E. coli transcript and the appropriate amount oftagged priming oligonucleotide and terminating oligonucleotide). TargetCapture Protocol. First there was prepared a microtiter plate containing200 .micro.L of wash reagent (Plate 2). Another microtiter plate (Plate3) for conducting amplification reactions was prepared, with each wellto be used for a reaction containing 30.micro.L of amplificationreagent. All three plates (Plates 1-3) were loaded into the magneticparticle processor unit. Magnetic beads harboring nucleic acid complexeswere isolated from Plate 1, washed in Plate 2, and then transferred intoPlate 3 using standard procedures familiar to those having an ordinarylevel of skill in the art. Plate 3 was removed from the magneticparticle processor unit, covered with an adhesive tape seal, and thenplaced into the temperature-controlled real-time instrument.

Real-Time Amplification Protocol. Plate 3 was incubated at 42.deg.C for5 minutes in the real-time instrument. The microtiter plate was removedfrom the real-time instrument and placed onto a 42.deg.C thermomixer.Each reaction well received a 10.micro.L aliquot of enzyme reagentcontaining detection probe, and was then covered with an adhesive tapeseal. The plate was shaken gently for 60 seconds on the thermomixer, andthen placed back into the real-time instrument at 42.deg.C where realtime assay monitoring was commenced. TTime values, which served asindicators of the amount of amplicon synthesized, were determined fromthe monitored fluorescence signals.

Results and Conclusion.

FIG. 21 graphically illustrates the benefits of the disclosed approachto nucleic acid amplification. Procedures that employed a tagged primingoligonucleotide complementary to a target of interest, a target capturestep, and a tag-specific priming oligonucleotide that was notcomplementary to the target of interest (i.e., the E. coli rRNA) yieldeddramatically reduced background amplification levels, and so easilypermitted discrimination between 0 and 1×10.sup.3 copies of thebacterial template nucleic acid. More specifically, the average TTimevalues determined for reactions carried out using 1×10.sup.5 copies,1×10.sup.3 copies, and 0 copies of the E. coli template were 24.7minutes, 30.6 minutes and 37.5 minutes, respectively. Taken togetherwith the results presented above, these findings were consistent withthe presence of bacteria-derived nucleic acids in common reagents usedfor conducting in vitro nucleic acid amplification reactions. Despitethis fact, the procedure employing a tagged priming oligonucleotide wasuseful for detecting E. coli nucleic acids contained in a test samplewithout interference from exogenous template nucleic acids contributedby the amplification reagents. For example, a qualitative assay fordetecting E. coli nucleic acids at a level of 10.sup.3 copies or greaterin a test sample could depend on achieving a threshold fluorescencesignal, or TTime value after a predetermined reaction time (e.g., 35minutes).

The following Example presents comparative results showing how twodifferent detection probes influenced the profiles of real-timeamplification run curves. The results further showed how the taggedpriming oligonucleotide approach could be used for discriminating 0 and10.sup.3 copies of the synthetic E. coli template nucleic acid—a levelapproximating the number of copies of 16S rRNA present in a singlebacterium.

Example 6 describes detection of E. coli rRNA templates in real-timeamplification reactions using three different detection probes.

Example 6 Alternative Torch Designs can Improve Assay Results

Amplification reactions were conducted and monitored in a real-timeformat using one of three different detection probes. The synthetictemplate nucleic acid, non-specific capture oligonucleotide, taggedpriming oligonucleotide, termination oligonucleotide, promoteroligonucleotide and tag-specific priming oligonucleotide used forperforming the reactions were identical to those used in the secondprocedure of the preceding Example. SEQ ID NO:14 is an exemplary taggedprimer oligomer comprising a target hybridizing region and a tag region.The target hybridizing region hybridizes to nucleobases 497 to 524 ofthe synthetic E. coli fragment of SEQ ID NO:13. This target hybridizingregion is also illustrated in SEQ ID NO:19. SEQ ID NO:10 is apromoter-based oligomer comprising a promoter region and a targethybridizing region. For the instant example, the 3′-end of this oligomeris blocked, thus SEQ ID NO:10 is a promoter provider oligomer. Thetarget hybridizing region for SEQ ID NO:10 hybridizes with nucleobases413 to 433 of the synthetic E. coli fragment of SEQ ID NO:13. Thistarget hybridizing region is also illustrated in SEQ ID NO:20. Fourreplicates were run for each condition. As before, detection probe wasadded with the enzyme reagent. Reagents and protocols for non-specifictarget capture, sample preparation, and real-time amplification alsowere essentially as described in the second procedure of the precedingExample. Notably, reactions were conducted using 0, 1×10′ or 1×105copies of the synthetic E. coli template. As above, 2′-O-methyl ribose(2′-O-Me) modifications of the polynucleotide backbone in the sequencespresented below are indicated by lower case “m.” Blocking moieties atthe 3′ termini of the promoter oligonucleotide and terminatingoligonucleotide comprised a 3′-to-3′ linkage that was prepared using3′-dimethyltrityl-N-benzoyl-2′-deoxycytidine, 5′-succinoyl-long chainalkylamino-CPG (Glen Research Corporation, Sterling, Va.; Cat. No.20-0102-01). Oligonucleotides, reagents and essential methods used inthe procedure were as follows.

TABLE 11 Oligonucleotides SEQ ID NO: Sequence 5′→3′ Comment 14GTTTGTATGTCTGTTGCTATTATGTCTACCTGCTGGCACGGAGTTA GCCGGTGCTTC 15GTTTGTATGTCTGTTGCTATTAT 10 ATTTAATACGACTCACTATAGGGAGAGAAGGCCTTCGGGTTGTAAAG-block 11 GmCmCmUmUmCmUmUmCmAmUmAmCmAmCmGmCmGm-block m = 2′-Ome16 KmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmTTT m = 2′OmeAAAAAAAAAAAAAAAAAAAAAAAAAAAAA K = g or t/u 17¹CmGmAmGmCmAmAmAmGmGmUmAmUmUmAmAmCm²GmCmUmCmGm³ m = 2′-Ome1 = fluorescein 2 = C9 linker 3 = DABCYL 18¹CmGmAmGmCmAmAmAmGmGmUmAmUmUmAmAmCmUmUmUmAmCm m = 2′-OmeUmCm²GmCmUmCmGm³ 1 = fluorescein 2 = C9 linker 3 = DABCYL 13See sequence listing Synthetic E.coli fragment

Reagents and Other Protocol Information: Reagents and experimentalprotocols were essentially as described under Example 5, with a slightchange to the conditions used for target capture. Non-Specific TargetCapture Protocol: Target Capture Reagent (TCR) Preparation. A stocksuspension of magnetic beads was mixed at room temperature for 25minutes. A 150.micro.L aliquot of the magnetic bead suspension was addedto 5 mL of TCR diluent (15.micro.g beads/reaction when using 50.micro.L/sample), and then slowly mixed at room temperature for 25minutes. Next, the non-specific capture oligonucleotide was added to 5mL of the TCR mixture to yield a final concentration of 0.12pmol/.micro.L. The prepared TCR was mixed gently at room temperatureuntil needed. Sample Preparation. Amplification solutions were preparedusing primerless AMP Reagent, promoter oligonucleotide and tag-specificpriming oligonucleotide. The prepared amplification solutions were mixedby vortexing and then maintained at 2-8.deg.C until needed. EnzymeReagents containing the molecular torch detection probes were nextprepared and maintained at 2-8.deg.C until needed. Dilutions of thetemplate rRNA were prepared in 0.2% LLS, as described above. Aliquots(50.micro.L) of the magnetic bead target capture solution weretransferred into the wells of a microtiter plate for a KINGFISHER 96(Thermo Fisher Scientific, Inc.; Waltham, Mass.) magnetic particleprocessor. Samples of diluted template, tagged priming oligonucleotideand terminating oligonucleotide were then added to 1.5 mL of 50%Transport Medium diluted with water. The target-containing samplemixture was vortexed, and 150.micro.L aliquots transferred into themicrotiter plate (Plate 1) wells containing 50 .micro.L target capturesolution (each well contained 0, 1×10.sup.3 or 1×10.sup.5 copies of theE. coli transcript and the appropriate amount of tagged primingoligonucleotide and terminating oligonucleotide).

Target Capture Protocol. The microtiter plate (Plate 1) was incubated at60.deg. C. for 15 minutes using a SOLO HT incubator (Thermo Labsystems;Franklin, Mass.). The microtiter plate was then placed on the bench atroom temperature and allowed to equilibrate for 5 minutes (Plate 1).Next, there was prepared a second microtiter plate containing200.micro.L of Wash Reagent (Plate 2). A third microtiter plate (Plate3) for conducting amplification reactions was prepared, with each wellto be used for a reaction containing 30.micro.L of amplificationreagent. All three plates were loaded into the magnetic particleprocessor unit. Magnetic beads harboring nucleic acid complexes wereisolated from Plate 1, washed in Plate 2, and then transferred intoPlate 3 using standard procedures familiar to those having an ordinarylevel of skill in the art. Plate 3 was removed from the magneticparticle processor unit, covered with an adhesive tape seal, and thenplaced into the temperature-controlled real-time instrument.

Real-Time Amplification Protocol. Plate 3 was incubated in the real-timeinstrument at 42.deg.C for 5 minutes. The microtiter plate was removedfrom the real-time instrument and placed onto the 42.deg.C thermomixer.Each reaction well received a 10.micro.L aliquot of Enzyme Reagentcontaining detection probe, and was then covered with an adhesive tapeseal. The plate was shaken gently for 60 seconds on the thermomixer, andthen placed back into the real-time instrument at 42.deg.C wherereal-time assay monitoring was commenced. TTime values, which served asindicators of the amount of amplicon synthesized, were determined fromthe monitored fluorescence signals.

Results and Conclusion: The results presented in Table 12 summarize theaverage TTime values (column 3), and the standard deviations of theaverage TTime values (column 4) for reactions conducted using thedifferent detection probes. The tabular summary confirmed that all ofthe tested detection probes yielded very good results in the real-timeassays. Each probe advantageously gave a very low signal at the 0 copylevel of input target. More specifically, amplicon detected in reactionscarried out using 0 copies of input synthetic template was essentiallyundetectable when the reactions included the detection probes of SEQ IDNO:17 and SEQ ID NO:18. These detection probes have target hybridizingregions configured to respectively hybridize to residues 462 to 477 andresidues 455 to 477 of SEQ ID NO:13. Reactions that included one of thedetection probes identified by SEQ ID NO:17 and SEQ ID NO:18 gaveoutstanding results that easily permitted detection of template nucleicacids corresponding roughly to the amount contained in a singlebacterium.

TABLE 12 Use of Alternative Detection Probes for Improved AssayDiscrimination Template Detection AvgTTime SDTTime Amount Probe (min)(min) 0 copies SEQ ID NO: 17 N/A N/A 10.sup.3 copies 38.2 2.81 10.sup.5copies 26.4 0.32 0 copies SEQ ID NO: 18 N/A N/A 10.sup.3 copies 35.92.33 10.sup.5 copies 28.8 0.45

Taken in view of the results presented Examples 5 and 6, each of SEQ IDNOS:12, and 17-18 represent preferred molecular torches for detecting E.coli using the methods described herein. Highly preferred probes usefulfor detecting E. coli nucleic acids will have target-complementarysequences corresponding to nucleotide positions 2-24 contained withinthe probe of SEQ ID NO:12 (i.e., a target hybridizing sequencesubstantially corresponding to SEQ ID NO:21), or nucleotide positions2-17 contained within the probe of SEQ ID NO:17 (i.e., a targethybridizing sequence substantially corresponding to SEQ ID NO:22), ornucleotide positions 2-24 contained within the probe of SEQ ID NO:18(i.e., a target hybridizing sequence substantially corresponding to SEQID NO:23). Generally speaking, probes useful for detecting E. colinucleic acids will have target hybridizing sequences of at least 16contiguous nucleotides contained within the sequence ofTGCGGGTAACGTCAATGAGCAAAGGTATTAACTTTACTC (SEQ ID NO:24). Overallpreferred lengths of desirable probes will be up to 39 nucleotides, morepreferably up to 29 nucleotides, more preferably up to 23 nucleotides,or still more preferably up to 16 nucleotides. Of course, useful probesmay include RNA and DNA equivalent bases, and include the complements ofthe foregoing described probes.

While the present invention has been described and shown in considerabledetail with reference to certain preferred embodiments, those skilled inthe art will readily appreciate other embodiments of the presentinvention. Accordingly, the present invention is deemed to include allmodifications and variations encompassed within the spirit and scope ofthe following appended claims.

1. A preannealing reaction mixture consisting essentially of aninactivatable target capture oligomer comprising at least a binding pairmember, a target hybridizing region comprising a nucleotide sequence ofat least 15 to 30 nucleobases in length and a tag-closing regioncomprising a nucleotide sequence of at least 3 to 20 nucleobases inlength, wherein said binding pair member, said target hybridizing regionand said tag-closing region are joined as a single molecule, and whereinsaid tag-closing region nucleotide sequence is substantiallycomplementary to a sequence contained within said target hybridizingregion. 2.-17. (canceled)
 18. A method for the specific hybridizationand capture of a target nucleic acid, said method comprising the stepsof: a. treating a target nucleic acid sequence in a nucleic acid samplewith an inactivatable target capture oligomer, wherein saidinactivatable target capture oligomer comprises a target hybridizingregion that is from about 15 contiguous nucleobases in length to about30 contiguous nucleobases in length, a binding pair member and atag-closing region, wherein said tag-closing region is from about 3contiguous nucleobases in length to about 20 contiguous nucleobases inlength, is substantially complementary to a portion of said targethybridization region, and does not stably hybridize to said targetnucleic acid, and wherein said target hybridizing region, said bindingpair member and said tag-closing region are joined as a single molecule;b. providing a set of conditions wherein the stringency of saidconditions biases said target hybridizing region towards stablyhybridizing with said target nucleic acid and not towards stablyhybridizing with said tag-closing region; c. providing a change in saidset of conditions wherein said stringency is reduced, allowinginactivatable target capture oligomer that is not stably hybridized witha target nucleic acid to form an inactive configuration by hybridizingsaid tag-closing region with said target hybridizing region; and d.performing a capture step wherein a complex comprising saidinactivatable capture oligomer stably hybridized with said targetnucleic acid in step b is captured. 19.-40. (canceled)
 41. A method forthe selective amplification of a target nucleic acid in a mixture ofnucleic acids comprising target nucleic acid and one or both of anon-target nucleic acid and a contaminating nucleic acid, said methodcomprising the steps of: a. selectively hybridizing and capturing atarget nucleic acid from a nucleic acid sample by a process comprisingthe steps of: (i). treating a target nucleic acid sequence in a nucleicacid sample with an inactivatable target capture oligomer, wherein saidinactivatable target capture oligomer comprises a target hybridizingregion that is from about 15 contiguous nucleobases in length to about30 contiguous nucleobases in length, a binding pair member and atag-closing region, wherein said tag-closing region is from about 3contiguous nucleobases in length to about 20 contiguous nucleobases inlength, is substantially complementary to a portion of said targethybridization region, and does not stably hybridize to said targetnucleic acid, and wherein said target hybridizing region, said bindingpair member and said tag-closing region are joined as a single molecule;(ii). providing a set of conditions wherein the stringency of saidconditions biases said target hybridizing region towards stablyhybridizing with said target nucleic acid and not towards stablyhybridizing with said tag-closing region; (iii). providing a change insaid set of conditions wherein said stringency is reduced, allowinginactivatable target capture oligomer that is not stably hybridized witha target nucleic acid to form an inactive configuration by hybridizingsaid tag-closing region with said target hybridizing region; and (iv).performing a capture step wherein a complex comprising saidinactivatable capture oligomer stably hybridized with said targetnucleic acid in step (ii) is captured; b. performing an amplificationreaction to generate an amplification product; and c. detecting saidamplification product to determine the presence or absence of saidtarget nucleic acid. 42-48. (canceled)
 49. The method of claim 41,wherein said target nucleic acid is detected in a probe-based detectionstep.
 50. The method of claim 41, wherein said tag-closing regioncomprises a nucleotide sequence that contains one or more nucleotideresidues selected from the group consisting of: an abasic nucleotideresidue, a wobble nucleotide residue, a mismatched nucleotide residuerelative to said residue's corresponding position in said sequencecontained within said target hybridizing region, and combinationsthereof.
 51. The method of claim 41, wherein said tag-closing regioncomprises a nucleotide sequence of at least 6 to 14 nucleobases inlength.
 52. The method of claim 41, wherein said tag-closing regioncomprises a nucleotide sequence of at least 6 to 9 nucleobases inlength.
 53. The method of claim 41, wherein said tag-closing regioncomprises a nucleotide sequence of 7 nucleobases in length.
 54. Themethod of claim 41, wherein said tag-closing region comprises anucleotide sequence of at least 6 to 9 nucleobases in length and saidtarget hybridizing region comprises a nucleotide sequence of 17nucleobases in length.
 55. The method of claim 41, wherein saidtag-closing region is joined to an end of said target hybridizing regionthat is opposite the end of said target hybridizing region to which saidbinding pair member is joined.
 56. The method of claim 55, wherein saidtag-closing region is joined to said end of said target hybridizingregion using a non-nucleotide linker.
 57. The method of claim 41,wherein said target hybridizing region is joined to an end of saidtag-closing region that is opposite the end of said tag-closing regionto which said binding pair member is joined.
 58. The method of claim 57,wherein said target hybridizing region is joined to said end of saidtag-closing region using a non-nucleotide linker.
 59. The method ofclaim 57, wherein said target hybridizing region is joined to said endof said tag-closing region using a nucleotide linker thereby forming acontiguous nucleotide sequence consisting essentially of said targethybridizing region and said tag-closing region.
 60. The method of claim59, wherein said binding pair member is a substantially homopolymericnucleotide sequence and is joined to said end of said tag-closing regionusing a nucleotide linker thereby forming a contiguous nucleotidesequence consisting essentially of said binding pair member, saidtag-closing region and said target hybridizing region.
 61. The method ofclaim 60, wherein said inactivatable target capture oligomer has a 3′ to5′ orientation such that the 5′ end of said binding pair member isjoined to the 3′ end of said tag-closing region and the 5′ end of saidtag-closing region is joined to the 3′ end of said target hybridizingregion.
 62. The method of claim 41, wherein step a.(iv) comprisesproviding a solid support material consisting essentially of acomplementary binding pair member; and providing conditions that allowfor binding of said binding pair member of said inactivatable captureprobe with said complementary binding member.
 63. The method of claim62, wherein said solid support material is a paramagnetic bead andwherein said complementary binding pair member is covalently attached tosaid paramagnetic bead.
 64. The method of claim 63, wherein saidcomplementary binding pair member is an immobilized probe and saidbinding pair member of said inactivatable capture probe is apolynucleotide region consisting essentially of a nucleotide sequencethat is substantially complementary to said immobilized probe.
 65. Themethod of claim 62, further comprising a washing step wherein saidcaptured target nucleic acid from step d. is retained while one or moreof non-captured nucleic acids, non-target nucleic acids, contaminatingnucleic acids, inactivated inactivatable target capture oligomer,reagents and sample debris are separated from said captured targetnucleic acid.
 66. The method of claim 62, wherein said solid supportmaterial is an amine, imine or guanidine coated paramagnetic bead.